Combined thesis 1

Analysis of in vivo Glycine transporter 1 functions by

transgenic approaches

Dissertation

zur Erlangung des Doktorgrades

der Naturwissenschaften

vorgelegt beim Fachbereich 14

Biochemie, Chemie und Pharmazie

der Johann Wolfgang Goethe-Universität

in Frankfurt am Main

von

Deepti Lall

aus Delhi (India)

Frankfurt 2011

(D30)

Die vorliegende Arbeit wurde in der Abteilung Neurochemie am Max-Planck Institut für

Hirnforschung in Frankfurt am Main unter Anleitung von Prof. Heinrich Betz durchgeführt

und vom Fachbereich Biochemie, Chemie und Pharmazie der Johann Wolfgang Goethe-

Universität in Frankfurt am Main als Dissertation angenommen.

Dekan: Prof. Dr. Dieter Steinhilber

1. Gutachter: Prof. Dr. Alexander Gottschalk

2. Gutachter: Prof. Dr. Heinrich Betz

Datum der Disputation:

A father's goodness is higher than the mountain,

A mother's goodness deeper than the sea.

For Mumma and Papa

INTRODUCTION

1 INTRODUCTION

Page | 5

INTRODUCTION

1.1 Inhibitory neurotransmission in the CNS

Communication between the neurons is a chemical process. When a neuron is

stimulated, the electrical signal (action potential) travels down the axon to the axon

terminals and triggers a series of chemical changes. Upon stimulation, there is an influx of

the Ca++ ions into the neuron which initiates the release of the neurotransmitters. These

neurotransmitters can then fulfill different functions in the brain, mainly either excitation

or inhibition of the postsynaptic neuron. Excitatory neurotransmitters act to stimulate

the postsynaptic neuron whereas Inhibitory neurotransmitters tend to block the changes

that cause an action potential to be generated in the responding cell.

GABA and Glycine are the major rapidly acting inhibitory neurotransmitters in the

brain. GABA is ubiquitously present in the CNS and therefore GABAergic inhibition is the

most common form of inhibition in brain. Glycine, in contrast is the major inhibitory

neurotransmitter in the caudal regions of the brain especially in spinal cord and brain stem

where it is crucial for regulation of motorneuron activity. Like GABA, glycine also inhibits

neuronal firing by gating Cl- channels but with a characteristically different pharmacology.

Page | 6

INTRODUCTION

The loxP sites are palindromic except for a 8-bp asymmetric core sequence that

provides each loxP site with an orientation (Hoess et al., 1986). If two loxP sites lie in the

same orientation on the same DNA strand, cre recombinase will catalyze the recombination

between the loxP sites and thus lead to a specific deletion of the flanked DNA segment. (Fig.

3.2).

Page | 7

Fig.3.3: Role of Cre recombinaseCre recombinase, often abbreviated to Cre, is a type I topoisomerase from P1 bacteriophage that catalyzes site-specific recombination of DNA between loxP sites (red triangles) (B). When Cre recombinase (red circle) is introduced, either as a transgene by crossing into a mouse line carrying the targeted gene locus or on a viral vector, the DNA between the loxP sites (red triangles) is removed, thereby inactivating the gene (C).Adapted from Rosenthal and Brown, 2007.

MATERIALS AND METHODS

2 MATERIALS AND METHODS

Page | 8


2.1 MATERIALS

2.1.1 Animals, Cell lines and Bacterial strains

Table 2-1: List of strains used in the study

Strain Source

Mouse (Mus musculus, C57/Black6) Charles River, Sulzfeld, Germany

Synapsin cre mouse line Zhu et. al. (2001)

GFAP cre mouse line Marino et. al (2000)

Emx cre mouse line Slezak et. al (2008)

Escherichia coli XL1-Blue StratageneTM (Amsterdam, Netherland)

E. coli DH5- InvitrogenTM (Carlsbad, USA)

Sv129/OlaHasd (E14TG2A) ES cell line Ulrike Mueller, Max Planck Institute for Brain Research, Frankfurt.

MTK-neo CD1 transgenic mice ref

Page | 9


2.1.2 Chemicals and plastic materials

All chemicals, unless otherwise stated, were ordered from the following companies:

Applichem (Darmstadt, Germany), Bio-Rad (Munich, Germany), Biotrend (Cologne,

Germany), Calbiochem Merck Biosciences (Schwalbach, Germany), Difco Laboratories

(Detroit, USA), Eppendorf (Hamburg, Germany), Fluka (Buchs, Switzerland), GE Healthcare

Biosciences (Freiburg, Germany), Gibco-BRL (Karlsruhe, Germany), Invitrogen (Carlsbad,

USA), Merck (Darmstadt, Germany), New England Biolabs (Ipswich, USA), Roche

Diagnostics (Mannheim, Germany), Roth (Karlsruhe, Gemany), Serva (Heidelberg,

Germany) and Sigma-Aldrich (Munich, Germany). All solutions were prepared with Milli-Q

water (Millipore, Wartford, USA).

All plastic materials were ordered from the following companies: Falcon (Le Pont De Claix,

France), Perbio Sciences (Bonn, Germany), Roth (Karlsruhe, Germany), Greiner (Darmstadt,

Germany), and Eppendorf (Hamburg, Germany).

2.1.3 Enzymes & others

All restriction enzymes were purchased from New England Biolabs, (Frankfurt am

Main, Germany) and Roche (Mannheim, Germany). The PAN Script DNA polymerase used in

PCR reactions was obtained from PANTM Biotech GmbH, (Aidenbach, Germany). The

Hotstart polymerase for genotyping inGlyT1 transgenic mouse tails was obtained from

Qiagen (Hilden, Germany). Ampli Gold taq polymerase from Applied Biosystems

(Darmstadt, Germany) was used for genotyping Glycine transporter 1 knockout mice, T4

DNA Ligase was obtained from Fermentas GmbH (St. Leon-Rot, Germany) and Proteinase K

and protease inhibitor cocktail were obtained from Roche (Mannheim, Germany).

2.1.4 DNA standard

Page | 10


As a reference marker for DNA, the Smart Ladder-Marker from Eurogentec (Cologne,

Germany) was used according to manufacturer’s instructions. This standard contains DNA

fragments of the following sizes: 10, 8, 6, 5, 4, 3, 2.5, 2, 1.5, 1, 0.8, 0.6, 0.4 and 0.2 kbp.

2.1.5 Protein standard

As reference for protein gels the SeeBlue2-Marker from Invitrogen (Carlsbad, USA)

was used according to manufacturer’s instructions. This marker contains the following

proteins: Myosin (250 kDa), Phosphorylase (148 kDa), BSA (98 kDa), Glutamate

Dehydrogenase (64 kDa), Alcohol Dehydrogenase (50 kDa), Carbonic Anhydrase (36 kDa),

Myoglobin Red (22 kDa), Lysozyme (16 kDa), Aprotinin (6 kDa) and Insulin (-chain) (4

kDa).

2.1.6 Membranes and films

Nitrocellulose membranes with a pore size of 0.45 µm from GE Healthcare were used

for southern blot analysis. For neutral transfer, Hybond-N+ and for alkaline transfer

Hybond-XL membranes were used. For Western blot analysis, nitrocellulose membrane

from GE Healthcare and ProtranTM from Schleicher and Schuell GmbH (Dassel, Germany)

were used. Autoradiographic films were purchased from BIOMAX MR (Kodak, Cedex,

France) or HyperfilmTM MP (A. Hartenstein, Erlangen, Germany) and GE Healthcare.

2.1.7 Commercial Kits for molecular biology

For standard molecular biology experiments, commercial kits were employed and all

the protocols were performed according to the manufacturer’s instructions.

Table 2-2: Commonly used kits.

Name Description Company

Plasmid kits Purification of plasmid DNA Qiagen, (Hilden, Germany)

Page | 11


(Mini, Midi, Maxi)

QIAquick Gel extraction kit Extraction of DNA from agarose gels

Qiagen

PCR purification kit Purification of PCR products Qiagen

SuperSignal Western Blotting chemiluminescent substrate kit

Enhanced chemilumescent (ECL) substrate for horseradish peroxidase (HRP) enzyme that permits low picogram detection of proteins in Western blot applications.

Pierce Thermo Scientific, (Schwerte, Germany)

QuickHyb Rapid hybridization solution

Used for quick hybridization of the radio-labeled probe to the nitrocellulose membrane

Stratagene,TM

Page | 12


2.1.8 Culture media and solutions

The following list of culture media and solutions were used during various cell-

biological and biochemical studies.

2.1.8.1 Bacterial Culture medium

Table 2-3: Culture medium used for growing bacteria

Name Composition

LB medium 1 % (w/v) NaCl, pH 7.5, 1 % (w/v) peptone , 0.5 %

(w/v) yeast extract

LB Agar LB medium with 1.5 % (w/v) agar

The LB or selection media were prepared by adding antibiotics in the required

concentrations to the autoclaved media, after it had cooled down to a temperature of ~55

°C. The final concentrations of the different antibiotics are listed in 2.1.8.2.

2.1.8.2 Antibiotics

Table 2-4: Antibiotics used for bacterial culture

Antibiotic Stock solution Final concentration

Ampicillin 100 mg/ml in water 50 µg/ml

Kanamycin 50 mg/ml in water 50 µg/ml

Tetracycline 10 mg/ml in 50 % ethanol 12.5 µg/ml

All antibiotics were sterile-filtered and stored at -20 °C.

Page | 13


2.1.8.3 Cell culture media for mammalian cells

Table 2-5: Cell culture media

Name Composition

DMEM medium +++

10 % (v/v) FCS, 100 µg/ml Pen/Strep (5000 U/ g/ml)μ , 2 mM L-Glutamine in 500 ml DMEM (Invitrogen).

Freezing medium 500 ml DMEM medium +++, 10 % (v/v) DMSO

Gelatin 0.5 % Gelatin in sterile distilled H2O

100 X poly-D-ornithine

150 g/ml, Poly-D-ornithine-hydrobromide in sterile distilled Hμ 2O

100 X poly-D-lysine

250 µg/ml Poly-D Lysine in distilled H2O

Page | 14


2.1.9 Medium and supplements for ES and MEF cell culture

Table 2-6: Cell culture media for ES and MEF cells

Name Composition Company

MEF medium DMEM, 10 % (v/v) FCS, 100 µg/ml Pen/Strep stock (5000 U/µg/ml), 2 mM L-Glutamine

DMEM and FCS Gibco-BRL

2X MEF freezing medium

MEF medium, 20 % (v/v) DMSO

ES cell medium DMEM, 10 % (v/v) FCS, 100 µg/ml Pen/Strep (5000 U/µg/ml), 2 mM L-Glutamine,10 mM NEAA, 0.1 mM -Mercaptoethanol, 1000 U/ml LIFβ

LIF (10,000 U/ml) :Life Technologies, (New York, USA).

ES cell selection medium

ES cell medium, 1000 U/ml LIF, 0.2 mg/ml Geneticin

Geneticin :Invitrogen

ES cell freezing medium

ES cell medium, 50 % (v/v) DMSO

Page | 15


All the media were sterilized through filtration with a 0.22 µm Bottle Top Filter and stored

for no longer than 2 weeks at 4 °C.

2.1.10 Electroporation of ES cells

For the electroporation of the ES cells the following media was used.

Table 2-7: Electroporation medium for the embryonic stem cells

Name Composition

10X HBSS 1X

1 M HEPES 20 mM

-Mercaptoethanolβ 0.1 M

1M NaOH 1 mM

Page | 16


The electroporation medium was prepared fresh for each round of electroporation and

sterilized through filtration with a 0.22 µm Bottle Top Filter (Becton Dickinson).

Page | 17


Page | 18


2.1.11 General buffers and solutions

Page | 19


All solutions were prepared in milliQ water from Millipore or as indicated. The

solutions were either sterile-filtered with a 0.45 µm Bottle Top Filter or autoclaved.

Page | 20


Table 2-8: Solutions for molecular biology

Page | 21


Page | 22

Name Composition

10X PBS 80 g NaCl, 2 g KCl, 14.4 g Na2HPO4, 2.4 g KH2PO4, pH 7.3

PMSF 100 mM in isopropanol

10X DNA loading buffer 20 % (v/v) Ficoll 400, 0.1 mM EDTA, 1 % (w/v) SDS, 0,25 % (w/v) Bromophenolblue, 0,25 % (w/v) Xylene Cyanol FF

20X SSC buffer 3.0 M NaCl, 0.3 M Trisodium citrate pH 7.0

50X Denhardt’s solution 1 g BSA, 1 g PVP, 1 g Ficoll

50X TAE 242 g Tris base, 57.1 ml glacial acetic acid, 37.2 g Na2EDTA.2H2O, pH∼8.5

Brain P2 prep membrane preparation medium

0.33 M sucrose, 1 mM EDTA, 1 mM PMSF, 10 mM HEPES-Tris pH 7.4

DNA extraction “salting out” buffer 200 mM NaCl, 100 mM Tris-Cl pH 8.5, 5 mM EDTA, 0.2 % (w/v) SDS, 210 µg/ml Proteinase K (25 µg/µl stock solution)

DNA isolation Buffer PI 50 mM Tris-Cl, 10 mM EDTA pH 8.0, RNase A (100 µg/ml)

DNA isolation Buffer P2 200 mM NaOH, 1 %(w/v) SDS

DNA isolation Buffer P3 3 M Potassium acetate, pH 5.5

HEK293 cell lysis buffer 150 mM NaCl, 5 mM EDTA, 1 % (v/v) Triton-X 100, 0.25 % sodium deoxycholate, 0.1 % SDS, 50 mM HEPES-Tris pH 7.4, 1 tablet of Protease inhibitor complete (Roche Biosciences).

Krebs Henseleit medium 125 mM NaCl, 5 mM KCl, 2.7 mM CaCl2, 1.3 mM MgSO4, 10 mM Glucose, 25 mM HEPES-Tris pH 7.4

LacZ stain washing buffer 2 mM MgCl2, 0.01 % (w/v) sodium deoxycholate, 0.02 % (v/v) NP-40 in 1X PBS pH 7.3

LacZ staining buffer 5 mM Potassiumferrocyanide, 5 mM Potassiumferricyanide, 0.5 mg/ml X-Gal (in DMF) in 1X PBS pH 7.3

LacZ fixative for embryo staining 1 % formaldehyde, 0.2 % glutaraldehyde, 5 mM EGTA, 0.02 % NP-40 in 1X PBS

LacZ staining buffer for embryo staining

5 mM Potassiumferrocyanide, 5 mM Potassiumferricyanide, 2 mM MgCl2, 01 % Na deoxycholate, 0.02 % NP-40, 1 mg/ml X-Gal (in DMF) in 1X PBS pH 7.3

Lysis buffer ES cells for PCR 100 mM Tris-Cl pH 8.5, 5 mM EDTA, 0.2 % (w/v) SDS, 0.2 M NaCl

Lysis buffer for ES cells 100 mM Tris-Cl pH 8.5, 5 mM EDTA, 0.2 % (w/v) SDS, 200 mM NaCl, 62.5 µg/ml Proteinase K


2.1.12 Solutions for protein biochemistry

Table 2-9: Composition of solutions

2.1.13 Solutions for immunocytochemistry and immunohistochemistry

Table: 2.10: Composition of solutions

Name Composition

2-4% PFA solution Add 2-4 g of PFA to 50 ml of sterile distilled water. Add a few drops of 1 N NaOH and heat at 55 °C till PFA dissolves. Add 10 mL of 10X PBS and make up the final volume to 100 ml with sterile distilled H2O.

Blocking and permeabilisation solution 1 % (v/v) goat serum, 2.5 % BSA, 0.1-0.3 % Triton-X 100 in 1X PBS pH 7.5

Quenching solution 100 mM glycine in 1X PBS pH 7.5

Quenching solution for DAB staining 0.3-1 % H2O2 in 1X PBS pH 7.5

Page | 23

Name Composition

10X Tris-Glycine buffer 30.2 g Tris base, 144 g glycine

4X Laemmeli buffer 8-11 % (w/v) SDS, 4 % (v/v) glycerin, 0.04 % (w/v) Pyronin Y, 20 % (v/v) -mercaptoethanol, 250 mMβ Tris-Cl pH 6.8

Blocking solution for western blotting

5 % (w/v) skim milk powder, 0.05 % (v/v) Tween-20 in 1X PBS

Ponceau-staining solution 3 % (w/v) Glacial acetic acid, 0.3 % (w/v) Ponceau S

SDS PAGE running buffer 1X TG buffer, 0.1 % SDS, pH 8.8

Stripping buffer for Western blot 420 µl -mercaptoethanol added to 15 ml Tris-Cl pH 6.8, 24 ml SDS (10 %) (v/v), 81 ml milliQ water

Western blot transfer buffer 20 % Methanol, 0.01 % SDS in 1X TG buffer, pH 8.8


2.1.14 Vectors

Table 2-11: List of vectors used (in the study)

Name Origin/ Reference Antibiotic resistance

pBluescript II SK (+) Stratagene (Heidelberg, Germany)

Ampicillin

pBK CMV Stratagene (Heidelberg, Germany)

Kanamycin

pcDNA3.1 Invitrogen, (Carlsbad, USA) Ampicillin

pcDNA3.1/ DLC myc Petra Scholze, MPIH, Frankfurt Ampicillin

pCCALL2-IRES-EGFP/anton Lobe et.al. (1999) Ampicillin

pCCALL2-IRES-EGFP/anton/GlyT1

Chigusa Shimizu Okabe (this study)

Ampicillin

pEGFP-C3 Clontech (Heidelberg, Germany)

Ampicillin

pPGKcrebpA Klaus Rajewsky, (Harvard Medical School, Boston, MA, U.S.A.)

Ampicillin

Page | 24

http://www.sciencedirect.com/science?_ob=RedirectURL&_method=outwardLink&_partnerName=27983&_origin=article&_zone=art_page&_linkType=scopusAuthorDocuments&_targetURL=http%3A%2F%2Fwww.scopus.com%2Fscopus%2Finward%2Fauthor.url%3FpartnerID%3D10%26rel%3D3.0.0%26sortField%3Dcited%26sortOrder%3Dasc%26author%3DRajewsky,%2520Klaus%26authorID%3D14827192400%26md5%3Dccbc1fa097bcd7a59448509b281ac581&_acct=C000032322&_version=1&_userid=616145&md5=2915b4f36030112071449db3eb42db8a


2.1.15 Antibodies

Table 2-12: List of primary antibodies used (in the study)

Name Antigen Species Dilution

WB

ICC/IHC

Source

mAb4a hybridoma supernatant

AS 96-105 from GlyR 1α

mouse Dr. Carmen Villman, Uni-Erlangen

GFP GFP isolated directly from Aequorea victoria

rabbit 1:1000 1:500 Invitrogen

VIAAT AS 75-87 from VIAAT rabbit 1:1000 Synaptic Systems, Göttingen

myc 9106 EQKLISEENL rabbit 1:200 Abcam, Cambridge, U.K

-Gal 4761β Full length native protein (purified from E. coli).

rabbit 1:250 Abcam, Cambridge, U.K

GlyT1(M7/M14)

rabbit 1:1000 Eulenburg et.al., 2010

GlyT2N N terminal cytoplasmic domain of rat GlyT2

rabbit 1:2000 Gomeza et.al., 2003a

GRP75 (JG1) 2799

AS 615-633 of Mouse Grp75

mouse 1:1000 Abcam, Cambridge, U.K

Page | 25


Table 2-13: Fluorescent conjugated secondary antibodies used for ICC and IHC

Name Dilution Source/Reference

Alexa 488-anti-mouse 1:500 Molecular Probes (Eugene, USA)

Alexa 546-anti-mouse 1:500 Molecular Probes (Eugene, USA)

Alexa 488-anti-rabbit 1:500 Molecular Probes (Eugene, USA)

Alexa 546-anti-rabbit 1:500 Molecular Probes (Eugene, USA)

Table 2-14: Peroxidase-linked secondary antibodies for western blot

Name Dilution Source/Reference

HRP-anti-mouse 1:10000 Dianova (Hamburg, Germany)

HRP-anti-rabbit 1:10000 Dianova (Hamburg, Germany)

Page | 26


2.2 METHODS

2.2.1 Biochemistry and molecular biology methods

2.2.1.1 Growth conditions and culture methods for microbial culture

The microbial cultures were grown under standard conditions as described (Bertani,

1951; Rose, 1990; Sambrook et.al., 1989). The E. coli strains were grown O/N in liquid LB

containing the appropriate antibiotics at 37 °C on an orbital shaker at 180-200 rpm. The

final concentrations of different antibiotics that were used for growing the cells are

described in 2.1.8.2.

2.2.1.2 Preparation of E. coli chemical competent cells

E. coli XL1-Blue cells were spread on agar plates containing 10 µg/ml tetracycline

and incubated O/N at 37 °C. Next day, a single colony was inoculated in a pre-culture of 5 ml

LB medium and incubated O/N at 37 °C with constant shaking. On the following day, the

O/N culture was transferred to 500 ml of pre-warmed LB medium and incubated at 37 °C at

300 rpm until the OD600 reached 0.5-0.7. The bacteria were then harvested by∼

centrifugation at 2600 x g, for 5 min at 4 °C. The cell pellet was resuspended in 8 ml of

sterile ice-cold 50 mM CaCl2 solution and kept for 1 hr at 4 °C. The cells were centrifuged at

1000 x g, 5 min at 4 °C and the pellet was carefully resuspended in 8 ml of sterile ice-cold 50

mM CaCl2 solution containing 40 % (v/v) glycerol. 200 µl aliquots of the bacteria were

frozen in liquid nitrogen and stored at –80 °C.

Page | 27


2.2.1.3 Preparation of E. coli electrocompetent cells

30 ml LB-medium was inoculated with a single colony of E. coli XL1-Blue bacteria

and incubated O/N at 37 °C at 200 rpm. 10 ml of the O/N culture was transferred next day

to 1 l of pre-warmed LB medium and incubated at 37 °C and 300 rpm until the OD 600

reached 0.5-0.7. The bacteria were then kept at 4 °C for 10-15 min and afterwards were∼

harvested by centrifugation at 3500 rpm, for 15 min at 4 °C. From here all the steps were

performed on ice using pre-chilled sterile solutions and glassware. The cell pellet was

subjected to three successive washes (resuspension in ice-cold ddH2O and subsequent

centrifugation at 3500 rpm for 15 min at 4 °C). Subsequently, the pellet was washed once

with 10 % (v/v) glycerol, and finally resuspended in 5 ml of 10 % glycerol. 100 µl aliquots

of the bacteria were frozen in liquid nitrogen and stored at –80 °C.

2.2.1.4 Preparation of bacterial glycerol stocks

After growing the bacterial culture in LB medium supplemented with appropriate

antibiotics, till the OD600 reached 0.5-0.7∼ , 800 µl of cell suspension was mixed with 800 µl

of sterile glycerol. The glycerol stocks were aliquoted, directly frozen in liquid nitrogen and

stored at -80 °C till further use. For revival of bacterial culture, 4-5 ml of LB medium was

inoculated by tiny amount of glycerol stock using an inoculation loop or autoclaved

toothpick. The inoculated LB medium was incubated O/N at 37 °C with gentle shaking.

Page | 28


2.2.1.5 Heat shock transformation of plasmid DNA

The microcentrifuge tubes containing 100 µl of chemical competent cells were

thawed on ice for 5 min before transformation. 10-50 ng of DNA were added to the

chemical competent cells and incubated on ice for 30 min. The transformation mixture was

heat-pulsed at 42 °C for 45 seconds and placed on ice for 2-3 min. 900 µl of pre-heated LB

medium (2.1.8.1) was added and incubated at 37 °C for 1 hr on a shaker at 200-225 rpm.

After recovery the bacterial cells were then plated on LB agar plates (2.1.8.1) containing the

appropriate antibiotic and incubated O/N at 37 °C.

2.2.1.6 Electroporation

Shortly before transformation, the E. coli electrocompetent cells were thawed on ice

for 5 min. The DNA was mixed with the cells and incubated additionally on ice for 20 min.

The bacteria-DNA mixture was added to the electroporation cuvette and placed in the

electroporation chamber of “Gene PulserII” (Bio-Rad, Munich, Germany) and electroporated

with the following settings: voltage of 2.5 kV, pulse controller low resistance 200 Ω, and

capacitance 25 µF. The cells were collected into 1 ml LB medium (2.1.8.1) and incubated on

a shaker at 37 °C for 1 hr. The bacterial culture was then plated on solid LB agar plates

containing appropriate antibiotics and incubated at 37 °C O/N.

2.2.2 Isolation and purification of plasmid DNA

2.2.2.1 Small-scale purification of plasmid DNA

The small scale plasmid DNA preparations were carried out using Qiagen’s Plasmid

mini kit (QIAGEN, Hilden, Germany). The DNA preparation is carried out by a modified

alkaline lysis protocol (Birnboim and Doly, 1979) followed by binding of the DNA to an

anion-exchange resin under appropriate pH and low-salt conditions and subsequent

washing and elution steps.

Page | 29


In brief, 5 ml LB medium containing the appropriate antibiotic were inoculated with

transformed E. coli XL1-Blue bacteria and incubated O/N at 37 °C, 200 rpm. Cells were then

pelleted at 5000 x g for 15 min and plasmid purification was performed following the

manufacturer’s instructions. The bound plasmid DNA was eluted from anion-exchange

resin using 50 µl low TE and stored at -20 °C.

2.2.2.2 Large scale plasmid DNA preparations ("midi/maxi preps")

Preparative purification of plasmid DNA was carried out using Qiagen’s Plasmid midi

kit or Plasmid maxi kits. In brief, a volume of 100 or 250 ml LB medium containing the

appropriate antibiotic was inoculated with transformed E. coli XL1-Blue bacteria and

incubated O/N at 37 °C and 200 rpm. Cells were then pelleted at 5000 x g for 15 min and

plasmid purification was performed following the manufacturer’s instructions. The eluted

DNA was resuspended in water or low TE to a final concentration of 1 µg/µl and stored at -

20 °C.

2.2.2.3 DNA extraction from mouse tissue

For the extraction of DNA from mouse tissue, approximately 3 mm of tails of

newborn mice were digested in a solution containing 350-500 µl of mouse tail lysis buffer

(2.1.11). For the brain samples, different regions of the brain were directly frozen on dry ice

after isolating them from mice and 350-500 µl of lysis buffer (2.1.11) was added to the

samples. The samples were incubated O/N at 56 °C and 800 rpm.

2.2.2.4 Phenol-chloroform extraction of the genomic DNA

The tubes containing the O/N digested samples were vortexed briefly and

centrifuged at 13000 rpm for 5 min in a tabletop centrifuge. The supernatant was

transferred to a new tube and an equal amount of mixture of

phenol:chloroform:isoamylalcohol (25:24:1) was added. The tubes were vortexed briefly

and centrifuged at 13000 rpm for 5 min. The upper aqueous phase was transferred to a new

sterile eppendorf tube and an equal amount of chloroform:isoamylalcohol (24:1) was

Page | 30


added. The tubes were vortexed briefly again and centrifuged at 13000 rpm for 5 min. The

supernatant was collected in a new eppendorf tube and 500 µl of iso-propanol was added to

the sample. Tubes were shaken and centrifuged again for 10 min at 13000 rpm to

precipitate the genomic DNA. The supernatant was discarded. The pellet was washed with

250 µl of 75 % ethanol, air-dried and resuspended in 100 µl of low TE buffer (2.1.11).

2.2.2.5 Genomic DNA isolation by “salting out”

Alternatively, the genomic DNA was isolated by salting out extraction. The tails were

digested overnight at 56 °C in lysis buffer for ‘salting out extraction’ (2.1.11). Tubes were

vortexed briefly and centrifuged at 13000 rpm for 5 min in a tabletop centrifuge. The

supernatant was transferred to a new tube and 150 µl of saturated NaCl was added to it.

The tubes were then incubated at 100 °C for 20 min and subsequently on ice for 15 min.

The sample mix was centrifuged for 15 min at 13000 rpm in a table top centrifuge at room

temperature. The supernatant was transferred to a new tube and 850 µl of 100 % ethanol

was added. Tubes were shaken and centrifuged for 10 min at 13000 rpm to precipitate

genomic DNA. The supernatant was discarded. The pellet was washed with 500 µl of 75 %

ethanol, air-dried, and resuspended in 100 µl of low TE buffer (2.1.11).

2.2.3 Analysis of the purified DNA

2.2.3.1 Agarose gel electrophoresis for the separation of the DNA fragments

For the separation of DNA fragments agarose gels ranging from 0.7 % to 3.0% were

used. The desired amount of agarose was dissolved in 1X TAE buffer by heating in a

microwave. After the agarose had cooled, ethidiumbromide (1 g/ml) was added to it. Theμ

warm agarose was slowly poured into a gel tank and combs were inserted. The set was kept

undisturbed till the agarose polymerized.

Page | 31


Then 1X TAE buffer was poured slowly into the tank till the buffer level stands 0.5-0.8 cm

above the gel surface. Wells were formed by gently lifting the combs. Standard DNA marker

(2.1.4) and DNA samples (containing glycerol and tracking dye) were loaded into the well.

After loading, the set up was connected to power supply and voltage was set to 120 V (or 8

to 10 V/cm length). The gel was run until the tracking dye reached ¾ of the gel length or

until the desired separation was achieved. For documentation, snapshots of UV

transilluminated gels were taken.

2.2.3.2 Determination of DNA concentration by spectrophotometry

According to the Lambert-Beer law the absorption of an aqueous solution of a

substance is directly proportional to its concentration, A=ε*c*l, where ε is the molar

extinction coefficient (M-1cm-1), c the concentration (M) and l is the path length of the light

through the sample (cm). The absorption is measured at 260 nm and 280 nm, which

represent the absorption maxima for nucleic acids and proteins, respectively. The

measured absorption is the sum of the absorptions of all the bases in the solution. The

concentration of the DNA solution was analyzed using Nanodrop ND-100 (Thermo Fisher

Scientific, Waltham, USA). The purity of the DNA preparation was estimated by calculating

the ratio of absorbances at 260nm and 280nm (A260/A280).

For RNA: A260/A280=2.0,

DNA: A260/A280=1.8 and

Protein: A260/A280=0.6.

A ratio of 1.8 - 2.0 is desired when purifying nucleic acids. If the ratio is less than 1.7, the

solution is probably contaminated by protein or phenol. The concentration of plasmid DNA

was analyzed and adjusted to a concentration of 1 µg/µl (1 OD260 = 50 µg/µl).

2.2.3.3 Extraction of DNA from agarose gels

Page | 32


To isolate the desired DNA fragments from a sample after restriction digestion or

PCR amplification, it was subjected to a gel electrophoresis (2.3.2.9). Under 302 nm

illumination, fragments of the desired size were cut out of the gel using a sterile scalpel and

placed in 1.5 ml micro-centrifuge tubes. DNA was purified with the QIAquick Gel Extraction

Kit (2.1.7) based on the principles of spin-column technology with the selective binding of

the DNA to silica-gel membrane. The purified samples were vacuum-dried and resuspended

in 10 µl H20.

2.2.3.4 DNA-sequencing

The separation, detection, and sequencing, of DNA products were performed by the

service provider MWG Biotech. Inc. (Ebersberg, Germany).

2.2.4 Enzymatic modification and manipulation of the DNA

2.2.4.1 Restriction digest of the DNA

Restriction endonucleases are enzymes that recognize specific sequences within

dsDNA and cut both DNA strands. Smith and Nathans (1975) discovered and characterized

the restriction endonucleases that are commonly used in the molecular cloning. These

enzymes cleave at specific sites within their recognition sequence that ranges from 4-8 bp

in length and is in most cases palindromic. The hydrolysis of both dsDNA strands can

generate 5’- protruding, 3’-protruding or blunt ends. The 5’ ends are always

phosphorylated and the 3’ends are hydroxylated. These characteristics make them a useful

tool in molecular biology for sequence specific fragmentation of DNA. One unit of the

restriction enzyme is defined as the amount of enzyme required to cut 1 g of DNA in 1μ

hour at 37 °C. For analytical DNA digests, usually 500 ng-10 g (for southern blot) of theμ

DNA was digested with 1-10 units of the corresponding enzyme at 37 °C for 1-2 hr. For

double digests involving enzymes requiring incompatible buffers, the DNA was digested

sequentially. Restriction enzymes were then inactivated at 65 °C for 20 min (for heat

Page | 33


sensitive enzymes) and the DNA fragments were isolated after electrophoretic separation

in an agarose gel using QIAQuick gel extraction kit.

2.2.4.2 Dephosphorylation of the DNA digested with restriction

Terminal 5’-phosphoryl groups can be enzymatically removed by treating dsDNA

with calf intestinal phosphatase (CIP), thereby preventing unwanted re-ligation of

restriction digested DNA. One unit of CIP is defined as the amount of enzyme required to

hydrolyze 1 M p-nitrophenolphosphate to nitrophenol in 1 min at 37 °C. To removeμ

5’phosphoryl groups to prevent re-ligation, 1-2 U of CIP was added to a heat inactivated

'restriction digest' and incubated for 30 min at 37 °C. Since CIP cannot be heat inactivated,

the treated DNA was subsequently gel purified using QIAquick gel extraction kit, (2.1.7).

2.2.4.3 Ligation of DNA fragments

The bacteriophage T4-encoded enzyme 'DNA ligase' catalyses the formation of

phosphodiester bonds between neighboring 3’-hydroxyl- and 5’-phosphoryl-termini. It

requires Mg2+ ions and ATP as co-factors. The efficacy of a ligation reaction is influenced by

several factors, e.g. incubation temperature, reaction volume and the concentration of DNA

termini. Addition of polyethylene glycol in the ligation buffer enhances the ligation efficacy

and reduces the incubation time. The enzymatic activity is measured as the ‘cohesive’ end

ligation unit', and is defined as the amount of enzyme required to achieve a 50% ligation of

HindIII digested λ-DNA in 30 min at 16 °C in 20 l reaction volume and a 5’ terminiμ

concentration of 0.12 M (300 g/ml).μ μ

DNA fragments were ligated by mixing 25-50 ng vector DNA with a threefold excess of

insert DNA. 0.5 l of T4-ligase and 1 l of 10 X ligation buffer were added and the reactionμ μ

mixture was brought to a final volume of 10 l with ddHμ 2O. The reaction was incubated O/N

at 16 °C. The reaction mixture was used directly for transformation without any further

purification.

Page | 34


2.2.5 DNA amplification: Polymerase chain reaction

The polymerase chain reaction allows the in vitro amplification of a specific DNA

sequence (Mullis et al., 1986). DNA synthesis starts at two primers that are flanking the

sequence to be amplified. One of the primers anneals to the sense and the other one to the

anti-sense strand of the amplicon. DNA synthesis is carried out in a thermocycler shifting

temperatures between 95 °C for the initial denaturation of the template, the specific

annealing temperature of the primers and 72 °C for the synthesis reaction. The DNA

polymerase of the thermophilic bacterium Thermus aquaticus (Taq polymerase) is routinely

used for the PCR which catalyzes the synthesis reaction at a temperature of 72 °C The

reaction mixture consists of a DNA template, specific primers to amplify the amplicon,

dNTPs and Taq polymerase with corresponding salt and optimal pH conditions. Usually, 24-

30 cycles are necessary to obtain sufficient quantities of the PCR product for subsequent

steps. For current project, Taq™ DNA polymerase (Invitrogen); PAN scriptTM polymerase

and Qiagen hotstart polymerase were used for different PCR reactions as listed Appendix II.

The PCR conditions used for different PCR reactions are described in Appendix III.

2.2.6 Random-labeling of DNA probes

DNA probes were radioactively labeled with -[Pα 32]-dCTP using the Random

Primed DNA Labeling Kit (Roche Applied Sciences). In brief, 10 ng – 3 µg of the DNA to be

labeled were denatured by incubating at 95 °C for 5 min. The DNA was centrifuged and then

subsequently used for labeling.

Page | 35


Table 2-15: Reaction mix for random labeling of DNA probes

Component Amount

Template DNA 9 µl (10 ng – 3 µg)

dTTP 1 µl

dATP 1 µl

dGTP 1 µl

Hexanucleotide buffer 2 µl

-[Pα 32]-dCTP 5 µl

Klenow enzyme 1.5 µl

Page | 36


The reaction was thoroughly mixed by pipeting and then incubated at 37 °C for 1 hr. After 1

hr, the reaction was stopped by adding 30 µl of STE buffer (2.1.11). The reaction mix was

vortexed and purified using G-50 columns (GE Healthcare Biosciences Freiburg, Germany).

The labeled probe was added to the column and centrifuged at 3000 rpm for 2 min. 1 µl of

the radiolabeled probe was used for measuring the radioactive counts. The probe was then

heated at 95 °C and added to the membrane containing the hybridisation buffer and

incubated at 42 °C O/N.

2.2.6.1 Southern blot analysis of genomic DNA

10 µg of genomic DNA was fully cut overnight with appropriate restriction enzymes

in a final volume of 35 µl. The samples were separated on 0.8 % 1X TAE agarose gel at 1.5

V/cm for 7 hr. The gel was treated sequentially with denaturation solution and

neutralization solution (2.1.11) and then transferred using neutral transfer, O/N onto a

nylon membrane (2.1.6) in 10X SSC. After the transfer, the DNA was crosslinked to the

membrane by UV irradiation (StratageneTM, UV crosslinker) and stored at -20 °C until

further use.

Hybridization reactions were performed either with QuickHyb hybridization solution

(StratageneTM) or hybridization solution (Table 2-16) in an oven equilibrated to the

appropriate temperatures. Blots were exposed at –70 °C using Kodak Biomax MR film

(2.1.6) with intensifying screens and developed after 3 days

Table 2-16: Hybridisation solution for Southern blot

Component Amount

Formamide 20 ml

Salmon sperm DNA* 1 ml

1M NaH2PO4, pH 6.5 2 ml

10 % SDS 2 ml

50X Denhardt’s sol

Page | 37


4 ml

0.5 M EDTA, pH 8.0 0.4 ml

20X SSC 12 ml

*Salmon sperm DNA was heated at 95 °C for 2-5 min and then pre-mixed with formamide

solution. All the rest components were added later.

2.2.7 Brain total RNA preparation

Different regions of mice brains were isolated and directly frozen on dry ice. Total RNA

from the brain samples was extracted using peqGOLD RNA islolation solution (PEQLAB

Biotechnologie GMBH, Erlangen, Germany) according to manufacturer’s instructions.

2.2.7.1 First strand c-DNA synthesis

cDNA synthesis is based on the characteristic feature of eukaryotic messenger RNAs

to a harbor defined polyadenylated tail on the 3′ end. The cDNA was mainly synthesized for

RT-PCR. Total RNA was mixed with oligo-dT primers. The amplification reaction was

carried out by Reverse TranscriptaseTM (Roche) at 50 °C.

Page | 38


In brief, 50 pmol of oligo-dT primer and 1 g total RNA were added to a nuclease-freeμ

microfuge tube. The mixture was heated at 65 °C for 10 min. 4 l of 5 X First-Strand Buffer,μ

0.5 l of Protector RNase inhibitor, 2 l of 10 mM dNTP’s and 0.5 l of reverseμ μ μ

transcriptase were added to the tubes and final volume of the mix was adjusted to 20 lμ

with ddH2O. This mix was incubated in the thermocycler with the following settings: 25 °C

for 10 min, 50 °C for 60 min. Finally the reaction was terminated by heating the reaction

mix at 85 °C for 5 min. The cDNA was then used as template PCR amplification. To obtain

pure cDNA which is required for PCR amplification, the mixture was incubated with 1 l (2μ

units) of E. coli RNase H at 37 °C for 20 min to remove >1kb RNA.

2.2.8 Cell biology methods

Page | 39


2.2.8.1 Coating of coverslips and culture plates with poly-D-ornithine and poly-D-

lysine

To facilitate the attaching of cells on the surface, sterile and ethanol-washed

coverslips (placed in 24-well-plates) and 6-well plates were incubated for a minimum of 2

hours with poly-D-ornithine or poly-D-lysine (15 µg/ml in PBS, pH 7.5) (Sigma, USA) at 37

°C and 5 % CO2. Before use, the coating solution was removed and wells were washed three

times with 1X PBS.

2.2.8.2 Culture and maintenance of HEK 293T cells

All steps were performed in a sterile hood with sterile solutions and media (MEM or

DMEM+++). Solution and media were preheated 30 min before use in a water-bath at 37 °C.

Twice per week, cells with a confluence of approx. 70-90 % were splitted. For splitting, cells

were washed once with 4 ml PBS. Cells were detached from the surface by addition of 1 ml

of preheated 0.25 % trypsin/EDTA (Gibco BRL). Finally, cells were resuspended in 5 ml of

the medium and plated on new 10 cm dishes containing 10 ml of the suitable culture

medium (2.1.9). Cells were cultured in a waterlogged incubator at 37 °C and 5 % CO2. After

30 culture passages, a new batch of cells was thawed.

2.2.8.3 Freezing and thawing of cell lines

Cells growing in a 10 cm dish with a confluence of approx. 80-90 % were washed

once with PBS (pH 7.5) and incubated for 1 min with 1 ml of 0.25 % trypsin/EDTA. The

cells were centrifuged at 800 rpm and resuspended in the culture medium. 1 volume of 2X

freezing medium (2.1.8.3) was added to the suspension. The cell dilution was then

transferred in 1 ml aliquots to Cryo-TubesTM (Thermo Fischer Scientific) and the tubes were

left overnight at -80 °C in a cryocontainer filled with isopropanol. Cells were stored in liquid

N2 until further use.

To revitalize frozen cells, a very rapid warming up of the tube in a water bath at 37 °C was

Page | 40


carried out. The thawed aliquot was mixed with 10 ml of prewarmed culture medium

(2.1.8.3) and distributed in 10 cm culture dishes. The cells were grown and passaged as

explained above in 2.2.8.2.

2.2.8.4 Transfection of HEK 293T cells using Lipofectamine 2000TM

HEK 293T cells were seeded on poly-D-ornithine-coated coverslips with culture

medium in the absence of antibiotic. Lipofection was carried out following manufacturer’s

instructions.

Briefly, a day before transient transfection, HEK 293T cells were seeded in 24-well (30,000

cells/cover-slip) or 6-well plates (50,000 cells per well) in antibiotic-free cell culture media

(2.1.8.2) On the next day for transfection of individual cover-slips, 2 µl of Lipofectamine

2000TM and 0.8 µg of plasmid DNA were separately incubated in 50 µl of DMEM medium for

5 min. For a 6-well plate, 4 µg of DNA with 10 µl of lipofectamine was used. After 5 min, the

solutions containing the DNA and lipofectamine were mixed and incubated at room

temperature for 20 min. Next the plasmid DNA with lipofectamine containing DMEM

medium was added directly onto the wells. For double transfection, 0.4 µg of the respective

plasmids (2 µg for a 6-well plate) were used for the same amount of the reaction mix as

described above. After 4 hr, culture dishes with transfected cells were supplemented with

500 µl or 10 ml of DMEM containing serum and antibiotics and further incubated for 12-24

hr at 37 °C and 5 % CO2 to allow the expression of the transfected constructs.

Page | 41


2.2.8.5 Preparation of lysates from HEK 293T cells

After 24-hr expression by HEK 293T cells, the cells were washed twice with PBS and

500 µl of HEK 293T cell lysis medium (2.1.11) was added to the plates. The cells were

scraped from the bottom of the culture plate using a cell scraper and transferred to a sterile

1.5 ml eppendorf tube. Finally, cell lysate debris was removed by centrifugation for 18000 x

g for 15 min. The supernatant was directly used for analysis or stored at -20 °C.

2.2.8.6 Immunocytochemistry on HEK 293T cells

Cells cultured on cover-slips coated with poly-D-ornithine or poly-D-lysine in 24-

well plates were washed once with ice-cold PBS and incubated with 0.5 ml of ice-cold 4 %

PFA solution (2.1.13) for 10 min on ice. The cells were then washed thrice with ice-cold

PBS. To quench unspecific fluorescence due to fixative procedures, cells were incubated for

10 min with 100 mM glycine in PBS followed by three subsequent washes with PBS. To

permeabilize cell membranes and block unspecific binding of the primary antibody, the

cells were incubated in 0.5 ml of blocking solution (2.1.13) for 1 hr at RT. Subsequently

incubation with the primary antibody was done (diluted in blocking solution, appropriate

dilutions are summarized in 2.1.15). 30 µl of the dilution was loaded onto a piece of

Parafilm (Pechiney, Chicago, USA) in a moist chamber to prevent drying of the solution.

Coverslips were then placed with cells facing down over the drop and incubated overnight

at 4 °C. On the next day, coverslips were washed with PBS for 10 min each. The cells were

then incubated with a fluorophore-linked secondary antibody diluted in blocking solution

(2.1.15) for 45 min at room temperature. The cover-slips were then washed thrice with

PBS, 10 min each. For the staining of cell nuclei, cells were incubated in DAPI solution

diluted 1:1500 in PBS for 10 min at RT in a dark chamber and again washed twice with PBS.

Cover-slips were mounted on microscope glass slides (76 x 26x 1 mm) (Paul Marienfeld,

Lauda-Königshofen, Germany) by placing them on a drop of Aqua Polymount (Polysciences

Inc. Warrington, USA) upside down. They were allowed to dry and stored at 4 °C until

further visualized.

Page | 42


2.2.8.7 Cryostat brain sectioning

For immunohistochemical analysis, mice were anesthetized by inhalation of

isofluoran (Deltaselect, Pfullingen, Germany) and killed by cervical dislocation. The head

was cut off with scissors; brain was carefully removed from the skull and rapidly frozen on

dry ice. The frozen brain was then embedded in Tissue-Tek (Sakura Finetek, Zoeterwoude,

Netherlands), placed on a cryostat holder and left at -18 °C to allow the embedding gel to

embed the tissue. Transverse or sagittal slices of 12-20 µm width were cut in a Cryostat

(Leica, Jung Frigocut, 2800E). 4-6 slices were transferred to coated microscope glass slides

(25x75x1.0 mm, SuperFrostR Plus, Menzel Glaeser, Braunschweig, Germany) and either

processed rapidly or stored for further analysis at -80 °C.

For checking EGFP expression, the brain was dissected out from the skull and washed

briefly with ice-cold PBS and pre-fixed in freshly prepared ice-cold PFA solution (2.1.13) at

4 °C overnight. The following day, pre-fixed brains were washed with ice-cold PBS for 15

min at room temperature. For cryoprotection, brain samples were transferred to a solution

of 10 % sucrose/ PBS and incubated for 4 hr at 4 °C. Next, they were transferred to 20 %

sucrose/ PBS and incubated O/N 4 °C. The following day, the solution was change to 30 %

sucrose/ PBS and brains additionally incubated O/N 4 °C. The brain was then dried

completely to exclude out any solvent. The brain were then embedded in Tissue-Tek

(Sakura Finetek, Zoeterwoude, Netherlands) and incubated on ice to allow the medium to

penetrate into the tissue. Finally, the embedded brains were rapidly frozen on dry ice and

stored at -80 °C till further use.

Page | 43


2.2.8.8 Immunostaining on brain sections

For immunostaining, tissue slides were dried at room temperature and fixed for 10

min in ice-cold 4 % PFA (2.1.13). Afterwards, slides were washed thrice for 10 min with

PBS to completely remove the fixative. Slides were then incubated in quenching solution

(2.1.13) for 10 min at room temperature. Membrane permeabilisation and unspecific

binding of the antibody was blocked through the application of blocking solution (2.1.13)

for 2 hr at room temperature. The slides were incubated in primary antibody (appropriate

dilutions in 2.1.15 in blocking solution) O/N at 4 °C in a moist chamber. Next day, unbound

antibody was washed out by washing thrice with PBS for 10 min each. The slides were

incubated with secondary antibodies (2.1.15) diluted in blocking solution for 45 min at

room temperature in a moist chamber. For the staining of cell nuclei, slides were incubated

in DAPI solution diluted 1:1500 in PBS for 10 min at room temperature in a dark chamber

and again washed twice with PBS. Following that, slices were mounted with Aqua

Polymount (Polysciences Inc. Warrington, USA).

2.2.8.9 Detection of -galactosidase in tissue sections (X-Gal staining)β

The bacterial enzyme β-galactosidase catalyzes the cleavage of the O1 bond of the

sugar β-D-galactose to a substituent. Due to broad substrate specificity the enzyme can also

be used to cleave organic compounds such as 5-bromo4-chloro-3-indolyl-β-D-galactoside

(X-Gal) giving rise to an indigo-colored precipitate (5-bromo-4-chloro-3-hydroxyindole)

under oxidizing buffer conditions. Furthermore, this enzyme can be expressed in

mammalian cells when placed under the appropriate regulatory elements and is usually

well tolerated. This property of the enzyme allows the use of β-galactosidase in transgenic

mice as a reporter for the detection of transgene activity or for the detection of promoter

activity of an endogenous gene in ‘knock-in’ approaches (Goring et al, 1987; Sanes, 1994).

Page | 44


To detect -galactosidase activity in transgenic mice, slides containing 14- mβ μ

vibratome sections were washed in LacZ washing solution (2.1.11) for 10 min at room

temperature. Slides were then washed in PBS thrice for 15 min each. Next, the slides were

placed in LacZ staining buffer (2.1.11) for 20 min to 24 hours at 37 °C in the dark. The

following day, sections were washed in PBS thrice for 15 min each which stopped the X-gal

reaction. The slides were mounted with Aqua Polymount. Tissue sections can be stored for

more than 1 year at room temperature in the dark without any detectable loss of tissue

integrity or diffusion of the indigo-colored precipitates.

2.2.8.10 Confocal microscopy, image acquisition and analysis

Cells were analyzed using Axioimager (Leica Microsystems, Bensheim, Germany).

Pictures were obtained at 1024x1024 pixel resolution using 20x, 40x and 63x magnification

objectives. The images were analyzed with Leica TCS-NT software. Merges were also

obtained using the same software. Images were further developed and organized by Adobe

Photoshop and Illustrator software (Adobe, San Jose, USA). Image quantification was

performed using ImageJ software (NIH, USA).

Page | 45


2.2.9 Embryonic stem cell (ES) culture

2.2.9.1 Culture of Embryonic stem cells

ES cells were routinely grown on a monolayer of MEF cells at 37 °C in a 5 % CO 2

atmosphere. Only low passages of ES cells were used (less than 15 passages), since longer

culture times adversely affect ES cell pluripotency. Cells were kept at a relatively high

density and the medium was changed every 24 hr. Cells were passaged when colonies

reached ∼60 % confluency. The plate was washed twice with prewarmed ES-HBSS,

incubated with ES trypsin/EDTA for 2 min at 37 °C in a 5 % CO2 atmosphere. The cell

aggregates were disrupted by pipetting with a P200 Gilson pipette five times and

subsequent incubation for 1 min at 37 °C. Trypsinization was stopped by adding ES culture

medium (2.1.9). The cell suspension was transferred to a 15 ml falcon tube and ES medium

was added to a final volume of 10 ml. The cells were pelleted for 5 min at 800 x g at room

temperature, resuspended in ES medium (with or without geneticin depending upon the ES

cells), and plated on a fresh MEF monolayer.

2.2.9.2 Thawing of ES cells

The vial containing the frozen ES cells was allowed to thaw rapidly in a 37 °C water

bath. When the cells were nearly thawed, they were transferred into a falcon tube

containing 10 ml of prewarmed ES cell culture medium. The cells were pipetted up and

down to break apart cell clumps. The cells were then centrifuged at 800 rpm, 22 °C for 5

min. The medium was carefully sucked off from the sides of the falcon and the pellet was

redissolved in 1 ml of prewarmed ES culture medium. The cells were then carefully seeded

on 6-well feeder-coated plates. The cells were allowed to settle down for 5 min at room

temperature and then carefully transferred to a cell culture incubator at 37 °C with an

atmosphere of 5 % CO2.

Page | 46


2.2.9.3 Preparation of mouse embryonic fibroblasts (MEF’s)

Pregnant CD1-MTK neo female mice were sacrificed at E13-E14 by cervical

dislocation after anaesthetization. The embryos were removed from the uterus, and after

cutting away the brain and dark red organs, were washed with fresh PBS in order to

remove as much blood as possible. The remaining tissue was minced with two scalpels,

cells/tissue were suspended in several ml of prewarmed trypsin/EDTA (about 1-2 ml per

embryo) and incubated with gentle shaking at 37 °C for 20 min. The suspension was

transferred to a falcon tube containing a suitable volume of prewarmed DMEM/10 % (v/v)

FCS and filtered through a nylon mesh (pore size 50 µm). The filtrate was subjected to low

speed centrifugation (800 rpm, 5min). The resulting cell pellet was resuspended in

prewarmed MEF medium and plated out at 1 embryo equivalent per 1 T75cm flask. The

flasks were precoated with 0.5 % gelatin before seeding MEF cells. The medium was

changed on the following day, with the fibroblasts being the only cells that attached to the

dishes. The fibroblast cultures were passaged for maximum 5 times before irradiation.

2.2.9.4 Feeder irradiation

Plates containing MEF cells were confluent within one to a few days after plating.

When the MEFs formed a confluent monolayer, they were trypsinized and replated in a T-

75 cm2 flask. When the flask was confluent, cells were trypsinized and either frozen in MEF

freezing medium or replated in 3 x T-75 cm2 flasks. After the cells had reached confluency,

the flasks were washed with HBSS, trypsinized, and splitted to a T-175 cm2 flask/each T-75

cm2 flask. Upon cell confluence, the feeders were trypsinized and pooled in 50 ml of MEF-

medium and treated with 3000 rads of gamma irradiation to inhibit cell growth and

division. The inactivated MEFs were then either frozen in MEF freezing medium (3 x 1 ml

aliquots per confluent T-75 cm2 flask) and stored at -80 °C or used for plating cell culture

plates for the culture of ES cells. For using the irradiated feeders, 1 vial of the feeders was

plated on a 0.5 % gelatin-precoated 1 x 6-well plate.

2.2.9.5 Freezing of cells

Page | 47


After trypsinization, ES cells or MEFs were pelleted for 5 min at 800 x g at room

temperature and resuspended in the appropriate volume of prechilled freezing medium (3

ml of MEF freezing medium for one confluent T-125 cm2 flask; 1 ml of ES freezing medium

for one confluent well of a 6-well plate). The cell suspension was transferred to prechilled

1-ml cryovials containing 500 µl of the respective freezing medium (2.1.9) and rapidly

frozen on dry ice or transferred quickly to a pre-cooled freezing device at -80 °C. The cells

were stored for 2 days to 2 weeks at –80 °C. For long-term storage, the cells were

transferred to liquid nitrogen tanks.

2.2.9.6 Electroporation of the transgene construct into ES cells

Plasmid DNA of the transgene vector was prepared according to the Plasmid maxi kit

manual and the DNA was linearized by restriction enzymes SfiI and Eam11051 to remove

the excess vector backbone. After digestion, the vector DNA was precipitated, washed twice

with sterile 75 % (v/v) ethanol, and then dissolved in electroporation buffer (2.1.10). All

steps were performed under sterile conditions using sterile consumables under the hood.

ES cells from 4 confluent wells of a 6-well culture plate were trypsinized with 0.25 %

trypsin/EDTA for 5 min at 37 °C. The reaction was stopped by adding 3 ml of prewarmed ES

cell medium. The cells were pipetted up and down to break apart any cell clumps. The cells

were then pelleted for 5 min at 800 x g at room temperature. The cell pellet was

resuspended in 500 µl of prewarmed sterile electroporation medium and transferred into a

0.4 cm electroporation cuvette (Biorad). The final volume of the suspension was made up to

1 ml including the amount of DNA to be added for the electroporation. 20 µg of linearized

DNA were added to the cell suspension and incubated for 5 min at room temperature. The

cuvette was then placed in the electroporation chamber (Biorad Gene Pulser) and

electroporated using the following settings: 500 µF, 240 V, = 5.7-6.0 ms. The cells wereτ

allowed to recover for 10 min at room temperature, transferred to 100 ml of prewarmed ES

culture medium, mixed and distributed equally into 10 cm dishes with feeder cells and

incubated at 37 °C with an atmosphere of 5 % CO2. After 24 hr, the ES medium was replaced

with ES cell selection medium containing geneticin. The ES cells were allowed to grow in

Page | 48


the selection medium for 5-7 days. The medium was exchanged regularly to allow proper

growth of the cells.

2.2.9.7 Selection of ES cell clones

One day before isolating ES cell colonies, 24-well plates with feeder cells were

prepared. The plates with electroporated ES cells were washed twice with 8 ml of

prewarmed HBSS. The cells were left in the second HBSS wash during picking. A P-200

pipette with 1-200 µl filter tips was used to pick individual colonies. First, the drug-

resistant ES cell colonies were slightly dislodged with the pipette tip containing 20 µl HBSS

and then transferred to a well of a 96-well plate. After picking 24 clones, 80 µl of

trypsin/EDTA was added to each well and the cells were incubated at 37 °C for 1 min. 10-20

pipetting cycles were used to generate single cell suspension by using a multichannel

pipettor. Then 80 µl ES cell selection medium was added to each well and mixed. The whole

volume of the solution was then transferred into 24-well feeder plates. The same procedure

was repeated until all clones in good shape had been isolated. Around 480 ES cell clones

were isolated and cultured.

2.2.9.8 Preparation of DNA from ES cells

500 µl of ES cell lysis buffer (2.1.9) was added to each well containing growing ES

cells in 24-well plate and incubated at 37 °C for 2-4 days. The lysate was transferred to a

microcentrifuge tube and 1 volume of isopropanol was added to precipitate the genomic

DNA. The DNA was then picked up with a pipette tip and transferred to a clean

microcentrifuge tube. The precipitated DNA was washed with 100 µl of 70 % EtOH. Tubes

were centrifuged at 1000 x g at room temperature and the precipitated DNA was dissolved

in 50 µl of low TE.

2.2.9.9 DAB staining of the ES cells

To check for the expression of EGFP and mycGlyT1 upon cre recombinase

expression, DAB staining was performed on ES cell clones. Selected ES cell clones were

Page | 49


grown on coverslips in 24 well plates till they reached confluence. To activate the transgene

in vivo, cells were electroporated with 15 µg of pGKCre plasmid and allowed to express for 2

days. The cells were fixed in 4 % PFA solution and treated with 0.3 % H2O2 in PBS for 30 min

to limit endogenous peroxidase activity. After washes with PBS, cells were blocked in

blocking solution (2 % BSA, 1 % NGS, 0.3 % Triton X) for 1 hr. For staining, cells were

incubated with rabbit anti-EGFP antibody for 1 hr. After 3 washes with PBS, cells were

incubated with anti-rabbit biotinalated secondary antibody for 1 hr. For DAB staining, cells

were incubated in a mixture of solution A and solution B (Vecstatin ABC kit, Vector

Laboratories, Burlingame, USA) for 2 hr at room temperature. For the DAB reaction, 1 tablet

of DAB (Sigma) was dissolved in 70 ml of 0.05 M Tris-Cl, pH 7.5 and filtered through a 0.45

µn filter. 3.5 µl of H2O2 was added to this solution and the coverslips were incubated in the

solution till the brown precipitate developed. The reaction was stopped by immersing the

coverslips in a solution of 0.05 M Tris-Cl, pH 7.5. For mounting the coverslips, they were

desiccated through a series of 70 %, 90 %, 100 % ethanol, and Xylene. The coverslips were

then mounted using . Respective non-cre electroporated ES cell clones were taken as

negative control for this experiment.

2.2.9.10 Preparation of ES cell clones for blastocyst injection

A week prior to injection, the frozen ES cells were thawed in 24-well plates with

feeder cells and expanded from 24-well to 6-well plates. The ES cells with small compact

colonies that were in logarithmic growth phase were selected for injection. On the day of

injection, the cell medium was changed and the cells were washed twice with prewarmed

HBSS. The cells were trypsinized by adding trypsin/EDTA. The cells were incubated at 37 °C

for 3-8 min to allow the dissociation of the cells from the plate. Trypsinization was stopped

by adding 1-2 ml of prewarmed ES cell selection medium and ES cell aggregates were

disrupted by pipetting 10 times. 7 ml of the prewarmed ES cell medium was added to make

a cell suspension. The cells were then centrifuged for 5 min at 1000 rpm and room

temperature. The supernatant was discarded and the cells were resuspended in 1 ml of

prewarmed ES cell medium. The suspension was then plated on gelatinized 6 cm plate

Page | 50


containing 4 ml of the ES cell medium. The cells were incubated at 37 °C for 45 min to allow

the feeders to sediment. The plate was then washed briefly with the supernatant and ES

cells plated on a fresh gelatinized 6 well plate. The cells were again incubated at 37 °C for

30 min. The plate was visualized under a microscope and if there were too much feeders

left, the above protocol was repeated for the third time. Finally, the plate was washed

briefly with the supernatant and the cell suspension centrifuged for 5 min at 1000 rpm and

room temperature. The cells were then resuspended in 1 ml of injection medium (ES

medium + LIF + 20 mM HEPES). The injection of the ES cells into the blastocyst was

performed by Frank Zimmerman at University of Heidelberg, Germany.

Page | 51


2.2.10 Glycine uptake experiments

2.2.10.1 Brain membrane preparation

Mouse brain samples were homogenized in 1 ml of ice-cold P2 prep membrane

preparation medium (2.1.11) using a Dounce-type glass/teflon homogenizer. The

homogenate was centrifuged (1000 x g, 5 min) at 4 °C. The pellet (P1) was discarded, and

the supernatant was centrifuged (17000 x g, 10 min) at 4 °C. The resulting pellet (P2) was

washed once with ice cold modified Krebs-Henseleit medium (2.1.11). The mixture was

centrifuged (17000 x g, 5 min) at 4 °C and the final resulting pellet was resuspended in ice

cold modified Krebs-Henseleit medium. Protein concentrations were determined using

Bradford protein assay system (Bio-Rad).

2.2.10.2 (3H) Glycine Transport Assay

20 µl aliquots of the membrane suspension (equivalent to 30–50 µg of protein) in 80

µl of Krebs-Henseleit medium were preincubated for 2 min at 37 °C. All experiments were

performed in triplicates Uptake was initiated by addition of 100 µl of modified Krebs-

Henseleit solution kept at 37 °C containing [3H]-glycine (2 µM) (1.52 GBq/µmol,

MovasekInstru Biochemicals, Brea, CA). After 1 min incubation with gentle agitation, uptake

was terminated by diluting the incubation mixture with 3 ml of ice-cold modified Krebs-

Henseleit medium followed by rapid filtration through a moistened filter (SM 111060, 45

µm pore size, Sartorius, Gottingen, Germany). Filters were rinsed twice with 3 ml of

modified Krebs-Henseleit medium. All dilution, filtration, and washing procedures were

performed within 15 s. Filters were dried and placed in microvials, and their radioactivity

measured by scintillation counting (Beckmann, Germany). The glycine mediated uptake

was calculated as follows

Relative uptake = cpm/ [total glycine concentration] (pmol) Total protein conc. (mg) * time (min)

2.2.11 Protein biochemistry methods

Page | 52


2.2.11.1 Protein determination by Bradford assay

The protein concentration of the samples was measured using the Bio-Rad ‘Bradford

Protein Assay’ The Bradford assay, a colorimetric protein assay, is based on an absorbance

shift of the dye Coomassie Brilliant Blue G-250 under acid conditions when a redder form of

the dye is converted into a bluer form on binding to protein. The binding of the protein

stabilizes the blue form of the Coomassie dye; thus the amount of the complex present in

solution is a measure for the protein concentration, and can be estimated by use of an

absorbance reading. The (bound) form of the dye has an absorption spectrum maximum

historically held to be at 595 nm. The increase of absorbance at 595 nm is proportional to

the amount of bound dye, and thus to the amount (concentration) of protein present in the

sample.

Due to presence of high lipid content in the membrane preparations, the protein

determination assay was modified. In short, in a microcentrifuge tube, 1 µl of the protein

sample was mixed with 49 µl of 0.2 N NaOH. 750 µl of H2O and 200 µl of the Bio-Rad reagent

were added to the mixture. The mixture was vortexed thoroughly. Subsequently the

absorption was read at the wavelength of 595 nm. The protein concentration could be

determined by reference to a curve. We used different dilutions of bovine serum albumin

(BSA) in 0.2 N NaOH as a reference control to plot the reference curve.

2.2.11.2 Discontinuous SDS Polyacrylamide Gel Electrophoresis

The most widely used denaturing and discontinuous polyacrylamide gel

electrophoresis (SDS-PAGE) method for protein separation was described by Laemmli

(Laemmli, 1970). In this method buffers of distinctive pH and composition generate a

discontinuous pH and voltage gradient in the gel. The discontinuity in pH and voltage

concentrates proteins in each sample into narrow bands thereby allowing the separation of

very dilute samples. The protocol primarily relies on denaturing proteins by heating in the

presence of SDS and β-mercaptoethanol (β-ME). Under these conditions, the subunits of

proteins are dissociated and their biological activities are lost. Most proteins bind SDS in a

Page | 53

http://en.wikipedia.org/wiki/Nanometre

http://en.wikipedia.org/wiki/Absorption_spectrum

http://en.wikipedia.org/wiki/Coomassie_Brilliant_Blue

http://en.wikipedia.org/wiki/Absorbance

http://en.wikipedia.org/wiki/Assay

http://en.wikipedia.org/wiki/Colorimetric


constant SDS-to-weight ratio, leading to identical charge densities for the denatured

proteins. Thus, the SDS-protein complexes migrate in the polyacrylamide gel according to

size, not charge. Most proteins are resolved on polyacrylamide gels containing from 5 % to

15 % acrylamide and 0.2 % to 0.5 % bisacrylamide. The detailed theory and protocol for

one dimensional gel electrophoresis has been described in Gallagher, 2006; Hames, 1990.

A glass plate and a 1mm spacer plate sandwich of the electrophoresis apparatus was

assembled according to Bio-Rad instructions. The separating gel solution of desired

percentage of acrylamide (8%-12%) was prepared freshly and poured along an edge of one

of the spacers until the height of the solution between the glass plates was 2/3rd of the

maximum height of the glass plates. The top of the gel was slowly covered with a layer (1

cm thick) of isopropanol. The gel was allowed to polymerize for 30 min at room

temperature. Once the gel had polymerized, the layer of isopropanol was poured off and

was twice rinsed with ddH2O to remove any residual isopropanol. The stacking gel solution

was freshly prepared and was poured slowly on top of the polymerized separating gel along

an edge of one of the spacers until the solution reached the top of the plates. A 1 mm Teflon

comb (10 teeth) was inserted into the layer of stacking gel solution. The stacking gel

solution was allowed to polymerize for 30 to 45 min at room temperature. After

polymerization, the teflon comb was removed carefully. After the comb was removed, wells

were rinsed with 1X SDS electrophoresis buffer.

For loading of the samples, 1/4 volume of SDS loading buffer (4X) was added to the protein

samples and incubated at room temperature (for analysis of membrane transporter

proteins) for 15 min with shaking. The samples were then centrifuged at 13000 rpm for 15

min. The centrifuged samples were then directly used for analysis. For analysis of soluble

proteins, the samples were heated at 95 °C for 5 min. 20-40 µg of the protein sample was

loaded per well. Electrophoresis was carried out at 25 mA per gel for 40 mins. 5 µl of See

Blue Plus2 marker (2.1.5) was used as molecular weight standard. After the loading buffer

had eluted out of the separating gel the power supply was disconnected. The gel was

carefully removed and subjected to protein blotting.

Page | 54


2.2.11.3 Western blot analysis

Western blot is an extremely useful tool for the identification and quantification of

proteins in complex mixtures of proteins that are not radiolabeled. In this technique,

electrophoretically separated protein samples (2.2.11.2) are transferred from a gel to a

membrane and probed with antibodies that react specifically with a particular antigenic

epitope. Proteins can be detected down to femtomole quantities, well below the detection

limit for most assay systems.

Following electrophoresis, the separated proteins were transferred from the SDS

polyacrylamide gel to the membrane (2.1.6) by electrotransfer using a Mini-Trans-Blot

electrophoretic chamber (Bio-Rad; Munich, Germany). The transfer was carried out in

transfer buffer (2.1.12) at 30 V O/N or at 100 V for 2 hr on ice. After the transfer, the

membrane was stained for 5 min in Ponceau solution to visualize the transferred protein

bands.

2.2.11.4 Immunodetection of blotted proteins

The specific identification of protein bands was performed by indirect

immunodetection. To avoid unspecific binding of the primary antibody, the membrane was

incubated in blocking solution (2.1.12) for 1-2 hr at room temperature with shaking. The

membrane was then incubated, either overnight at 4 °C or for 1 hr at 25 °C in blocking

solution containing the desired dilution of the primary antibody (2.1.15). After 3 washes

with 1X PBS/0.05 % (v/v) Tween-20 for 10 min each, the membrane was incubated with

the secondary antibody conjugated to HRP diluted in blocking buffer for 45 min at room

temperature. The blots were again washed thrice with 1X PBS/0.05 % (v/v) Tween-20 and

HRP coupled secondary antibody was directly detected using the SuperSignal West Pico

Chemiluminescent Substrate kit (Pierce, USA) following the manufacturer’s instructions.

HRP is an enzyme that catalyzes the oxidation of luminol-based substrate leading to an

excitation of the chemiluminescent substrate that generates light at the site of reaction,

which is visualized through exposure to an X-ray film. The membrane was then covered

Page | 55


with a transparent film and an ECL photographic film (Hyperfilm™, Amersham Biosciences)

was exposed to the membrane. The time of exposure varied from 1 sec to 15 min depending

on the signal intensity. Afterwards, films were developed by a Kodak X- OMAT 2000

processor (Kodak, Atlanta, USA).

Page | 56

RESULTS

3 RESULTS (PART I)

Page | 57

RESULTS

3.1 Role of Glycine and Glycine transporters in the CNS

Glycine is a major inhibitory neurotransmitter in the spinal cord and brain stem,

acting at strychnine-sensitive GlyR’s-chloride (Cl-) channels (Legendre, 2001). At these

glycinergic inhibitory synapses, the post-synaptic actions of glycine are terminated by a

rapid reuptake mechanism, which is mainly mediated by glycine transporters (GlyT’s).

GlyT’s which include GlyT1 and GlyT2, belong to the family of sodium/chloride dependent

transporters (Eulenburg et.al., 2005). GlyT1 is widely expressed in astrocytic glial cells

whereas GlyT2 is localized to the presynaptic terminals of glycinergic neurons

predominantly in brain stem and spinal cord (Zafra et.al., 1995 a,b).

Apart from acting as a classical inhibitory neurotransmitter, glycine is unique in that it also

acts along with glutamate, as an essential co-agonist of the excitatory NMDA receptors

(NMDAR’s). In higher regions of the brain such as hippocampus, glycine has been shown to

simultaneously increase Gly-R mediated inhibition and facilitate NMDAR-dependent

plasticity at excitatory synapses (Zhang et.al., 2008). The simultaneous activation of

excitatory NMDAR’s and inhibitory GlyR’s may provide a homeostatic regulation of

hippocampal network function.

In the hippocampus, GlyT1 is mainly expressed by astrocytes and neuronal GlyT1 is only

found at glutamatergic synapses (Cubelos et.al., 2005). At these synapses, it is postulated

that astrocytes via GlyT1 are the major source of hippocampal glycine (Yang et.al., 2003).

However, a direct evidence for the release of glycine via this transporter in still lacking. To

understand more about the regulation of glycine (release/or uptake) in more frontal

regions of the brain, I generated an inducible GlyT1 transgenic mouse line which is

described in this thesis.

Page | 58

RESULTS

3.1.1 Considerations for construct design for generation of an inducible

(in)GlyT1 transgenic mice

By “classical” definition, a transgenic organism carries extra, often foreign (i.e. from a

different organism) DNA into its genome called a transgene. This transgene can be

integrated into the host genome either randomly, via viral injection or by creating a site-

specific ‘knockin’.

In “classical transgenesis,” the DNA of interest is sub-cloned downstream of a suitable

promoter element that drives its expression, followed by a stop signal to stop transcription.

To a large extent, this promoter element determines the level, tissue specificity, and

temporal pattern of transgene expression. For e.g. for exerting control over the expression

of the transgene in a cell-type or tissue specific manner, tissue or cell-type specific

promoters are used, (Glial Fibrillary Acidic Protein (GFAP) promoter, specific for glial cells).

In contrast, for a more ubiquitous expression, ubiquitin promoter and -actin promoter areβ

more commonly used. Reporter genes encoding proteins, such as GFP, LacZ are cloned

downstream of the promoter as a read out for the promoter activity in a cell or a developing

animal. (Voncken and Hofker, 2005). Regulatory elements such as viral enhancers are also

commonly used in the transgenic constructs to enhance transgene expression and achieve

position-independent and copy number dependent expression of the transgene.

The problem encountered during “classical transgenesis” is that most of the animals show

high variation in expression pattern due to random insertion of the transgene in the

genome, including undesirable expression of the transgene in a particular tissue(s) (ectopic

expression). This could lead to phenotypes that nonspecifically affect the nature of the

system in question and can lead to results which might not be due to the transgene

influence. Another problem arises due to sensitiveness of the expression of the transgene to

its proximity to transcriptional activators and silencers. Silencer elements are distributed

throughout the genome, making it possible that some integration sites yield little or no

expression of the transgene.

Page | 59

RESULTS

To overcome the problems posed by classical transgensis, temporal control over transgene

expression is required. This is achieved by an inducible system wherein the expression of

the transgene can be turned on or off at defined times and positions.

“Conditional transgenesis” is used to have a better control over the (trans) gene expression

in a given organism and is integrated by the use of tissue-specific promoters and/or control

element usage (Voncken and Hofker, 2005). Due to this, transgene expression is confined to

a defined target tissue or selected cells within a tissue. Conditional transgenesis employs

the use of “binary switches” that can be used to turn “on” or “off” the transgene. Two types

of binary switch models are commonly used to conditionally induce the transgene.

a) Tetracycline controlled transgene expression: the Tet-system: this system is based

upon the tetracycline resistance operon from E. coli. Inducibility in this system

depends on the presence or the absence of tetracycline or its analog doxycycline. In

the “tet-off” system, presence of tet or doxycycline prevents the expression of the

transgene by effectively binding to the tTA. This is in contrast to the “tet-on” system

where the presence of tetracycline allows the expression of the transgene. Despite

its convenience for inducing transgene expression, this system suffers from some

disadvantages. Firstly, the requirement to combine two transgenic lines often leads

to unexpected patterns of transgene expression. In addition to regional variability, a

line may also show chimeric expression within a given region (Yamamoto et.al.,

2001). Another disadvantage of the system arises from the exposure of the animals

to tetracycline. To induce gene only in the adult animal requires the persistent

exposure (beginning at the conception) of the mice to tetracycline to prevent

transgene expression. This can be expensive and the effects of the long term

exposure to tetracycline are unclear. Also, conditional control of transgene

expression in Furthermore, tetracycline clears slowly away from the animal thereby

delaying transgene expression after long term exposure (Yamamoto et.al., 2001).

This makes this system unsuitable for the investigation of acute transgene effects.

Page | 60

RESULTS

b) Transgene induction by the removal of a transcriptional block: Cre/loxP system: This

system makes use of a recombination process derived from bacteriophage P1 as

explained in (cross-reference to the introduction). Here an on/off transgenic system

employs a silenced transgene, in which a strong transcriptional block is bracketed by

two similarly oriented loxP sites. The DNA sequence flanked by loxP sites is said to

be “floxed.” A second independent transgenic line expresses cre recombinase under

a tissue specific promoter. Cre recombinase-mediated recombination results in the

removal of entire floxed element, leaving one loxP site behind and thereby deleting

(knockout) or expressing (knockin) the region of interest. However, the expression

of the transgene is unidirectional i.e. once the expression is put “on” it can’t be put

“off.”

One obvious advantage of the Cre/loxP system lies in the fact that it can be used to generate

mice lacking a protein in a particular tissue to avoid early lethality or severe developmental

consequences. Furthermore, this system allows for a spatial and temporal control over

transgene expression and takes advantage of the inducers with minimal pleiotropic effects.

For these reasons we employed the use of this system for the generation of the inGlyT1

transgenic mice which would allow for the time and cell/ tissue specific activation of the

transgene. For the generation of the transgene construct, a ubiquitous promoter along with

a strong enhancer element was chosen which could constitutively and ubiquitously express

higher levels of the transgene in all cell types. A floxed reporter silencing cassette was

cloned downstream of the promoter to allow for the selection of the cells harboring the

construct. cDNA of the gene of interest was cloned downstream of the floxed cassette. A

second reporter was inserted downstream of the cDNA of interest to act as a marker for the

removal of the floxed cassette and the expression of the cDNA.

The inducible expression of the transgene construct works in the following way: the

introduction of the transgene into the host cells leads to the expression of the reporter in all

cell types under the ubiquitous promoter. This is being referred to as “silenced construct.”

Expression of cre recombinase under a cell or tissue specific promoter leads to the excision

Page | 61

RESULTS

of the floxed cassette and the expression of the transgene. This is referred to as “cre-

recombined construct.” Since GlyT1 is expressed upon cre excision, it is also referred to as

inGlyT1. A second reporter is used to detect the deletion of floxed cassette and the

expression of cDNA of interest. The Internal Ribosome Entry Site (IRES) element present in

the vector allows the bicistronic expression of the gene of interest and the second reporter

separately. IRES drive the translation of the cDNA of interest and reporter independently

such that both the proteins are expressed in the same cell. (Fig. 3.1).

Page | 62

RESULTS

Fig. 3.1: Strategy for the generation of inGlyT1 transgenic miceThe expression of GlyT1 in the transgene construct is inducible. The silenced construct expresses LacZ as a first marker prior to cre excision. Expression of GlyT1 is prevented by a poly-A signal downstream of LacZ/Neor cassette. The expression of GlyT1 is accomplished by the expression of cre recombinase which recognizes the two loxP sites in the construct and thus removes the silencing cassette. This is known as “cre-recombined construct.” EGFP is expressed as a second reporter marker after cre excision.

Page | 63

RESULTS

3.1.2 Generation of the transgene construct

The expression vector used for the generation of the inGlyT1 mouse line was based

on an approach from Lobe et.al., 1999 and

Please_Select_Citation_From_Mendeley_DesktopNovak et.al., 2000 The expression of the

transgene is driven by ubiquitously active chicken -actin promoter. An enhancer elementβ

cassette (CAG cassette) from Cytomegalovirus (CMV) (Xhu et.al., 2001, Niwa et.al., 1991) is

cloned upstream of the promoter to direct strong expression. Expression of the transgene

GlyT1 is prevented by the expression of a silencing cassette containing a first reporter, a

LacZ/Neor fusion protein; (Friedrich and Soriano, 1991) that was followed by a triple

repeat of the simian virus (SV40) polyadenylation signal. The reporter along with the stop

signal is flanked by two loxP sites. This allows the removal of the silencing cassette upon

expression of cre recombinase (Fig. 3.2, B). The original vector map is listed in appendix III.

Transgene construct was generated by cloning the GlyT1 cDNA downstream of the floxed

LacZ/Neor cassette via Bgl II and Xho I restriction sites in the vector (Fig. 3.2, A). An IRES

was inserted downstream of the GlyT1 cDNA to allow for bicistronic expression of EGFP

(Enhanced Green Fluorescent Protein) derived from the jellyfish, Aequorea victoria (Chalfie

et.al., 1994) and GlyT1. This construct was designated as iLacZ/GlyT1-EGFP.

Page | 64

RESULTS

Fig.3.2: Schematic drawing of iLacZ/GlyT1-EGFP construct used for generating transgenic mice (A). GlyT1 cDNA was cloned using Bgl II/Xho I restriction sites into the pCCALL2-IRES-EGFP/anton vector. (B) Transgene expression is driven by the ubiquitously active CMV-enhanced chicken ß-actin promoter. A floxed LacZ/Neor cassette serves as a selection marker and a silencing cassette due to a poly-A signal at its 3' end. Following that is coding sequence for GlyT1 and EGFP. LacZ reporter is used as first marker, expressed prior to Cre excision, and the EGFP reporter as the second marker, expressed after Cre excision.

For the ease of understanding the terms used in this study, the “silenced construct” will be

referred to as iLacZ/GlyT1-EGFP and the “cre-recombined” construct as i LacZ/GlyT1-Δ

EGFP Characterization of tagged GlyT1 constructs

Page | 65

RESULTS

In order to distinguish between the endogenously and transgenically expressed

GlyT1, a DNA sequence encoding for C-myc epitope was introduced into N-terminus (GlyT1-

N) and C-terminus (GlyT1-C) of the GlyT1 cDNA respectively (Fig. 3.3, A). The tagged GlyT’s

(mycGlyT1) were cloned into vector pcDNA3.1 and their functional properties were

analyzed by glycine uptake experiments and Western blotting (Fig. 3.3, B, and C) in HEK

293 cells.

Glycine uptake experiments were performed after 2 days as described in section 2.2.10.2.

The glycine uptake experiments revealed no significant difference between the uptake

activities of cells transfected with tagged and untagged GlyT1 constructs. Both tagged

constructs showed a concentration-dependent uptake of radiolabeled glycine, although

GlyT1-N showed much less uptake activity than GlyT1-C. Untransfected HEK 293 cells and

cells transfected only with pcDNA3.1 also showed minimal glycine uptake activity which

was much less than cells expressing the transporter constructs (Fig.3.3, B). Thus, it can be

concluded that the tagged transporter constructs were functional and can transport glycine.

For western blot analysis, lysates from HEK 293 cells transfected with different constructs

were prepared as described in section 2.2.8.5 and detected using anti-myc and anti-GlyT1

antibodies (Fig. 3.3, C). The cells expressing both myc-tagged constructs were recognized by

anti-myc and anti-GlyT1 antibodies respectively. When probed with anti-myc antibody,

immunoreactivity was detected for GlyT1-N as well as GlyT1-C whereas the untransfected

and cells expressing wildtype GlyT1 didn’t show any immunreactive bands. On probing

with anti-GlyT1 antibody, cells expressing both myc tagged constructs and endogenous

GlyT1 were recognized. The different band sizes at 110 kDa, 98 kDa and 58 kDa depict the

different complex glycosylated and unglycosylated forms of GlyT1 (Fig. 3.3, C). Since N-

terminally tagged construct showed glycine dependent uptake activity and was more

reliably detected in the western blots, it was further chosen for the generation of inGlyT1

transgenic mice.

Page | 66

RESULTS

Fig 3.3: Characterization of the tagged GlyT1 constructs(A) Depict the membrane topology of glycine transporter 1 with tags inserted at N and C terminus of the glycine transporter 1 (purple and red circles) respectively. (B) Depict 3 [H] glycine uptake activity by different constructs when expressed in HEK 293 cells. (C) Western blots analysis of membrane lysates expressing differently tagged constructs when probed with anti-myc and anti GlyT1 antibodies. (Original uptake and western blot data from Chigusa Shimizu Okabe).

3.1.3 Verification of the transgene construct

After the generation of the transgenic construct, different regions of it were verified

using PCR, restriction digest analysis and sequencing.

Page | 67

RESULTS

PCR was performed using primer pair 1S/2AS to check for the correct insertion of

mycGlyT1. The binding sites for the primers are listed in appendix I. The primer pair

resulted in a fragment of 727 bp from vector iLacZ/GlyT1-EGFP in which GlyT1 was cloned.

The corresponding original vector, pCCALL2-IRES-EGFP/anton which lacked GlyT1, didn’t

show any amplified product (Fig. 3.4, A). The amplified fragment was cut from the gel and

sequenced to check for the correct sequence of the fragment. The sequenced fragment was

verified by aligning it to the original GlyT1 cDNA sequence using Sequencher (Ann Arbor,

U.S.A) (data not shown). The sequencing confirmed the correct insertion of mycGlyT1 into

iLacZ/GlyT1-EGFP vector.

Different enzymes were chosen for restriction digest analysis upon prediction analysis by

the program MacVector (Cambridge, UK). Single as well as multiple base cutters were

chosen to digest the plasmid. The complete restriction map of the vector as well as

predicted fragment sizes is given in appendix IV. Single base cutter SfiI, linearized the

plasmid. (Fig.3.4, B). A double digest with SfiI/AhdI and SfiI/Eam11051 released a fragment

of 1273 bp. Multiple cutters ∼ ApaI, ClaI and SpeI also released expected size fragments.

However, digests with PstI and NotI were spurious and unpredicted fragment sizes were

obtained (Fig. 3.4, B). Restriction analysis with other enzymes gave fragments as predicted

by the restriction map (data not shown).

To check for the presence of functional loxP sites in the construct, the plasmid was treated

with cre recombinase in vitro. In brief, the plasmid iLacZ/GlyT1-EGFP was treated with cre

recombinase (NEB) in 1X cre recombinase buffer at 37 °C for 30 min. The reaction was

stopped by incubating the mixture at 70 °C for 10 min. The plasmid was then transformed

into E.coli chemocompetent cells. DNA was isolated from the colonies obtained after

successful transformation.

If the two loxP sites present in the vector are correctly recognized by cre recombinase, then

it would lead to excision of the region between the loxP sites as depicted in Fig. 3.4, C. Two

single base cutters XhoI and XbaI were chosen to distinguish between the “silenced

plasmid” and “cre-recombined” plasmid. The restriction site for XbaI is located before the

Page | 68

RESULTS

first loxP site and for XhoI after the second loxP site (Fig. 3.4, C). A “non-cre” recombined or

a “silenced construct” will release a fragment of 7538 bp upon an ∼ XhoI/XbaI digest.

However, if the loxP sites are recognized by the cre recombinase, the region between the

two loxP sites would be deleted and a digest with XhoI/XbaI will only release a fragment of

2567 bp.∼

As shown in Fig. 3.4, D, the restriction digest of the DNA isolated from transformed colonies

released a fragment of 2567 bp. Thus it can be concluded that the loxP sites present in∼

the iLacZ/GlyT1-EGFP vector were recognized by the purified cre recombinase which lead

to the recombination between the two sites, thereby excising the region in between them.

This implied for functional loxP sites in the vector which could be recombined in vitro.

Additionally, the transgene construct was sequenced using different primer pairs (as listed

in appendix I) and the acquired sequences were aligned using the software Sequencher

(Ann Arbor, U.S.A). The sequencing analysis showed a correct insertion of mycGlyT1 into

the vector. Different regions of the vector were also sequenced to check for any mutations

within the coding regions of different reporter regions and for the correct orientation of the

loxP sites (data not shown).

Page | 69

RESULTS

Fig. 3.4: Validation of the transgene constructThe plasmid iLacZ/GlyT1-EGFP was verified via PCR and restriction digest analysis. (A) Show the PCR fragments obtained from the vector lacking inserted mycGlyT1 and one that contains mycGlyT1. (B) The restriction pattern obtained by the digestion of plasmid iLacZ/GlyT1-EGFP with different restriction enzymes. (C) Vector map showing the position of the loxP sites and cutting sites for XhoI and XbaI. (D) Verification of the functional loxP sites in the plasmid.

Page | 70

RESULTS

3.1.4 Heterologous expression of the transgene construct in HEK 293 cells

After the transgene constructs was generated and verified, their functionality was

checked in HEK 293 cells. For the experiments, iLacZ/GlyT1-EGFP and I LacZ/GlyT1-EGFPΔ

plasmid DNA were transfected into HEK 293 cells (see 2.2.8.4) and the expression of the

markers, -gal and EGFP, were analyzed after 2 days of expression.β

To check for the cells expressing -gal encoaded by the LacZ gene in the construct, X-Galβ

staining of the transfected HEK cells was performed. X-Gal, also called as bromo-chloro-

indolyl-galactopyranoside, is an oragnic compound which acts as a substrate for the

enzyme -galactosidase. X-gal is cleaved by -galactosidase yielding galactose and 5-β β

bromo-4-chloro-3-hydroxyindole. The latter is then oxidized into 5, 5'-dibromo-4, 4'-

dichloro-indigo, an insoluble blue product. This blue color can then be visualized by naked

eye. The EGFP fluorescence was analyzed by fluorescence microscopy after PFA fixation of

the cells.

The untransfected HEK 293 cells did not show any blue color upon treatment with X-Gal or

any immunofluorescence (Fig. 3.5, A1 and A2). In contrast, the cells expressing

iLacZ/GlyT1-EGFP showed blue color due to the expression of the enzyme -gal (Fig. 3.5,β

B1). In the same cells no EGFP fluorescence was detected since the expression of EGFP was

silenced (Fig. 3.5, B2). Cells transfected with I LacZ/GlyT1-EGFP Δ did not show any -galβ

expression due to the loss of LacZ/Neor cassette upon cre recombination (Fig. 3.5, C1).

However, these cells showed EGFP fluorescence where the soluble EGFP was accumulated

uniformly all over the cell when visualized by fluorescence microscopy (Fig. 3.5, C2). These

results showed that the reporters and the stop element were functional in the transgene

construct and the LacZ/Neor silencing cassette could be removed by cre recombinase in

vitro.

Page | 71

RESULTS

Fig. 3.5: Functionality test of the constructs in HEK 293 cellsTo check for the functionality of the constructs, HEK cells were transfected with the indicated plasmids. Expression of -galβ or EGFP was analyzed by X-Gal assay or fluorescence microscopy. (A1, A2): untransfected HEK cells stained for -galβ and checked for EGFP fluorescence. (B1, B2): Cells transfected with iLacZ/GlyT1-EGFP show blue stained cells but no EGFP fluorescence. (C1, C2): HEK cells transfected with I LacZ/GlyT1-EGFP Δ , lacked -gal expression but show EGFP fluorescenceβ with the soluble EGFP accumulated all over the cell. Scale bar: 50µm

Immunostaining with different antibodies was also performed on fixed and permeabilized

transfected HEK 293 cells to check for the expression of the markers -gal, EGFP, andβ

mycGlyT1. Different dilutions of the antibodies which were used are listed in section 2.1.15.

To check for the expression of -gal, immunostaining with rabbit anti- -gal and Alexa 546β β

antibody was done. EGFP fluorescence was analyzed by fluorescence microscopy. The

Page | 72

RESULTS

untransfected HEK 293 cells did not show any stained cells when probed with the antibody

(Fig. 3.6, A1, and C1). The cells transfected with plasmid iLacZ/GlyT1-EGFP showed

expression of -gal localized both in the cytoplasm as well as in the cell membrane (Fig. 3.6,β

A2, white arrows). These cells, however, did not express EGFP (Fig. 3.6, A2, and C2). This

can be explained by the fact that in plasmid iLacZ/GlyT1-EGFP, only -gal is expressed andβ

the expression of EGFP is silenced due to the presence of LacZ/Neor silencing cassette.

However, cells transfected with plasmid I LacZ/GlyT1-EGFP did not show any -galΔ β

immunofluorescence due to in vitro excision of the silencing cassette (Fig. 3.6, A3), but

express EGFP (Fig. 3.6, B3, and C3, white arrows).

Fig. 3.6: Immunostaining of HEK 293cells with anti -Gal antibodyβExpression of the markers -gal and EGFP upon staining with anti -gal antibody. (A1-C1) Showβ β untransfected HEK 293 cells; (A2-C2) and (A3-C3) depict staining of HEK 293 cells expressing iLacZ/GlyT1-EGFP and I LacZ/GlyT1-EGFP respectively. (B1-B3): depict the cell nuclei co-stainedΔ with DAPI. Scale bar: 50 µm.

Page | 73

RESULTS

To analyze the expression of mycGlyT1 and EGFP, transfected HEK 293 cells were stained

with rabbit anti-myc antibody and anti-rabbit Alexa 546 (see 2.1.15). EGFP fluorescence

was analyzed by fluorescence microscopy as described previously.

In untransfected HEK 293 cells no myc immunreactivity was observed. Also, these cells did

not show any EGFP fluorescence or autofluorescence (Fig. 3.7, A1-D1). HEK 293 cells

transfected with plasmid iLacZ/GlyT1-EGFP also did not show any staining. This can be

explained by the fact that this plasmid contains the LacZ/Neor silencing cassette which

prevents the expression of mycGlyT1 and EGFP (Fig. 3.7, A2-D2). In contrast, the cells

transfected with plasmid I LacZ/GlyT1-EGFP show expression of mycGlyT1 as seen by theΔ

red channel (Fig 3.7, A3, white arrows). These cells also express EGFP (Fig 3.7, B3, white

arrows). Furthermore, it was observed that these two channels co-localize (Fig 3.7, C3,

white arrows), which meant that there was co-expression of both proteins in the same cell.

Together, these finding indicate that the stop element in the construct was functional since

there was no expression of mycGlyT1 and EGFP in cells transfected with iLacZ/GlyT1-EGFP.

Also, it proves that there is a bicistronic expression of both mycGlyT1 and EGFP since both

of these proteins can be detected together (Fig 3.7, D3, white arrows, yellow co-localized

dots) in the same cell transfected with I LacZ/GlyT1-EGFP.Δ

Page | 74

RESULTS

Fig. 3.7: Detection of mycGlyT1 and EGFPFluorescence images showing HEK 293 cells transfected with iLacZ/GlyT1-EGFP and I LacZ/GlyT1-ΔEGFP plasmids respectively. Staining for the myc antibody is shown in red channel and for EGFP in green. Co-localization is shown by yellow color. (A2-D2) show cells transfected with iLacZ/GlyT1-EGFP and probed for myc and EGFP respectively. (A3) cells transfected with I LacZ/GlyT1-EGFPΔ show expression of mycGlyT1 and EGFP (B3). The expression of mycGlyT1 and EGFP is in the same cells as depicted by the yellow co-localization (D3). Scale bar 50µm.

Page | 75

RESULTS

3.1.5 Backbone removal and linearization of construct

Before using the iLacZ/GlyT1-EGFP plasmid for the generation of the transgenic mice, a

part of its backbone was removed that was not necessary for the transgene functions. Two

restriction enzymes, SfiI and Eam1105 I were chosen which could delete the parts of the

plasmid which were not required. Restriction digest of the plasmid with these enzymes

removed parts of the vector backbone downstream of the second polyadenylation signal

(Fig. 3.8, A). Both these enzymes cut at single sites in the plasmid at positions 11225 bp and

12498 bp respectively

As depicted in the gel representative gel below, single restriction digest with both SfiI and

Eam1105 I linearized the plasmid iLacZ/GlyT1-EGFP (Fig. 3.8, B, lanes 2 and 3). However, a

double digest removes part of the vector backbone and releases a fragment of 1273 bp (Fig.

3.8, B, lane 4).

Fig. 3.8: Backbone removal and linearization of the plasmid iLacZ/GlyT1-EGFP(A) Shows the restriction map of the plasmid iLacZ/GlyT1-EGFP showing the location of restriction sites of the enzymes SfiI and Eam1105 I. (B) Representative gel showing the digestion of plasmid iLacZ/GlyT1-EGFP with SfiI and Eam1105 I.

Page | 76

RESULTS

3.1.6 Genetic manipulation of the Embryonic stem (ES) cells

For the generation of the transgenic mice, the E14 (129/OLA) embryonic cell (ES)

line was used. The use of ES cells over the conventional technique of using transgene

constructs for pro-nucleus injections for the generation of the transgenic mice has several

advantages. For eg:

a) Single copy integrations of the transgene construct are more likely to be obtained by

electroporation in ES cells as compared to pro-nucleus injection where multiple

integrations of the transgene are more common. Single copy integrations are more

advantageous over multicopy integration since silencing of the transgene can occur

if cells have more than one copy of the transgene in their genome. If using a cre/loxP

system for the generation of the transgenic mice, then the recombination between

multiple loxP sites can pose problems by causing cryptic recombination events in

cells harboring the multicopy integrations and the desired recombination may not

be obtained.

b) ES cell clones can be tested before the generation of the transgenic mice for the

suitable expression of the transgene.

c) Only few mouse lines carrying the desired integration are obtained as compared to

pronucleus injection where high numbers of mouse lines have to be screened to

check for suitably expressing lines. If the transgene has been integrated into the

germ cells of the blastocyst embryos, then its highly probable that the expression of

the transgene would be maintained over generations (germ-line transmission).

The only limiting factors for generation of the mouse line with help of ES cells are that the

process is time consuming and for the mice must show a germline transmission to allow the

expression of the transgene to be analyzed over generations.

To maintain the ES cells in an undifferentiated state, they were cultured on the feeder layer

of irradiated mouse primary embryonic fibroblasts (MEF) cells in the presence of

recombinant leukemia inhibitory factor (LIF) in the medium.

Page | 77

RESULTS

3.1.6.1 Isolation of ES cell clones

For the generation of the inGlyT1 mice, E14 ES cells (Fig. 3.9, A) were electoporated

with the linearized iLacZ/GlyT1-EGFP plasmid and grown for seven days under the

selection of G-418 (Fig. 3.9, B, and C). Since the LacZ/Neor fusion protein was expressed

under the control of the transgene promoter in the plasmid, G-418 resistant ES cell clones

were expected to express the transgene.

When the Neor ES colonies were large enough for expansion, those with undifferentiated

morphology were isolated and selected for further analysis. A total of 480 ES cell clones

were isolated out of which 400 survived which were then expanded. (Table 3.1).

Table 3.1: Summary of ES cell clones obtained

Description Number Percentage (%)

Total clones isolated 480

Surviving clones 400 83.33

Homogeneous clones 50 12.5

Non-homogeneous clones 250 62.5

Non-expressing clones 100 25

Once individual ES cell colonies were expanded, they were checked for the expression of the

transgene. X-gal staining of the expanded ES cells was performed to check for the

expression of the -gal. Some of the clones showed a homogeneous X-gal stain, while othersβ

were heterogeneous or didn’t express the transgene at all (Fig. 3.9, D). This could be

explained by the fact that the selection of the clones in the ES cell selection medium was not

complete. Since geneticin is utilized by the growing cells, they lower or deplete their

surroundings with the antibiotic and thus facilitate the growth of the non-resistant ES cells.

Page | 78

RESULTS

Fig. 3.9: Genetic manipulation of ES cells(A) Shows wild type E14 cells growing on a feeder layer of MEF. (B) Once the ES cells were expanding, they were electroporated with the transgene construct. (C) Depicts the electroporated Neor ES cell colonies. (D) X-Gal staining of the isolated ES cell clones showing the different kinds of the clones obtained.

Page | 79

RESULTS

3.1.6.2 Characterization of the ES cell clones

For all the ES cells clones isolated, they were expanded on a 24-well plate and

genomic DNA was isolated from them (See 2.2.9.8). GlyT1 specific genotyping PCR was

performed using primer pair 44S/44AS (see Appendix I) and GlyT1 (+) clones were

identified. A representative gel is shown in Fig. 3.10 with GlyT1 (+) ES cell clones showing a

fragment of 497 bp. 300 of the total ES cell clones which were isolated showed a GlyT1∼ ∼

specific band. Correspondingly, all the ES cell clones which were obtained were frozen (See

2.2.9.5) and stored in liquid nitrogen until further use.

Fig. 3.10: Characterization and freezing of ES cells(A) PCR showing ES cell clones positive for GlyT1. (B) The morphology of the GlyT1(+)ES cell clones prior to freezing. Most of the cells showed round morphology, a requisite for undifferentiated ES cell colonies.

Page | 80

RESULTS

3.1.6.3 Identification of the ES cells carrying a single copy integration of the

transgene: Multicopy PCR

In general, transgenes integrate at random sites in any of the chromosomes of the

genome of host cells. As a result, different cells may be expected to show integration of the

transgene at different chromosomal locations. The number of copies integrated per

genome ranges from one to several hundred. When multiple copies are integrated, they

are mostly integrated at one site joined to each other in either head-to-tail, head to head or

tails to tail fashion, i.e., as a concatemer. In a small proportion of cases, multiple copies can

also be located at several sites in the same genome. However, rarely single copy

integration of the transgene also occurs.

Since the transgene construct was electroporated into the ES cells, which then randomly

integrated into the ES cell genome, it was found imperative to identify the ES cell clones

which carried only a single copy integration of the transgene. This was essential since the

number of copies integrated into the host genome can influence the expression pattern of

the transgene dramatically. Multicopy integrations can give high levels of expression of the

transgene but the expression varies between different animals and a lot of animals need to

be analyzed to get any reliable data. This is circumvented in single copy integrations. A

PCR strategy was designed in a way to distinguish between the clones carrying single copy

and multicopy integrations.

For this PCR, primers were designed in a way which could differentiate between single

copy and multicopy integrates. Two primers were designed in a way that the first primer

binds at the beginning of the construct and the second primer before the first linearization

site. (Fig. 3.11, A). After PCR, different fragment sizes are expected depending upon the

orientation of the transgene and the formed concatemers. For single copy integration, no

PCR product is expected since two primers bind in opposite direction, thereby making no

amplification product (Fig. 3.11, B). The primers will yield a PCR product (products) if

there is/are multicopy integrations, depending upon the orientation of the integrations

(Fig. 3.11, C).

Page | 81

RESULTS

The different sizes expected for different multicopy integration are as follows:

Heat to Tail 910 bp

Head to Head 1028 bp

Tail to Tail 728 bp

ES cell clones which showed homogeneous LacZ stain were checked for their copy number.

In total, 9 clones were checked with the PCR. ES cell clone 12, 149, and 269 did not show

any amplification product upon PCR analysis. (Fig. 3.11, C). This implied that these clones

probably had single copy integration of the transgene. In contrast, clone 141 showed a

single band at 900 bp (Fig. 3.11, C **), which can be explained by the multicopy∼

integration of the transgene in a head to tail fashion. However, the pattern obtained with

clone 140 was more complex. Apart from showing a band at 900 bp (Fig. 3.11, C **), it also∼

showed a band at 700 bp, which might correspond to a tail to tail integration of the∼

transgene (Fig. 3.11, C ***). From this it can be deduced that this clone carried multicopy

integration of the transgene maybe at different chromosomal locations and in different

orientation. ES cell clones 11, 28, 29, 33 also did not show any band upon PCR amplification

implying that these clones also had single copy integration. However, clones 29 and 33

were further excluded from the study since during expansion, the cells did not show

homogeneous LacZ stain and became undifferentiated upon prolong culture.

Page | 82

RESULTS

Fig. 3.11: Multicopy PCR: Identification of the clones carrying single copy integration of the transgene(A) Representative diagram of the transgene construct depicting the primer binding sites. The dotted square box indicates the region before the first linearization site. (B) Shows the different types of multicopy integration possible and the product size expected for each configuration. (C) Gel showing different ES cell clones on which the multicopy PCR was performed. The red star (*) indicate clones having single copy integration and then used for further analysis. (**) and (***) indicate bands for the clones which had multicopy integration. The other faint bands which are visible in the gel picture are unspecific bands which were also present in wild type ES cells (data not shown).

Page | 83

RESULTS

3.1.6.1 Molecular test to check for in vitro cre activation of the transgene: “Non-

activation/Activation PCR”

After ES cell clones containing single copy integration of the transgene were

identified, it was decided to “activate” the transgene in vitro i.e. to excise the silencing

LacZ/Neor cassette upon cre expression. For this, ES cells clones carrying the “silenced

transgene” (i.e. cells electroporated with transgene construct iLacZ/GlyT1-EGFP) and a

single copy integration were expanded on 2*24-well plates. These cells were called as “-

cre-recombined” clones. To induce cre recombination in the expanding ES cells, a transient

expression of a plasmid carrying cre recombinase was carried out. The pGKCre plasmid

(Ref.) was electroporated into half of the expanding ES cells and allowed to express for two

days. pGKCre plasmid contains a bacteriophage P1 recombinase cre with a SV40 large T

antigen nuclear localization signal (NLS-Cre) under a Phosphoglycerate kinase I promoter

(PGK) promoter,which allows the expression of cre recombinase in mammalian cells. The

cells treated with pGKCre were called as “+ cre-recombined” clones. Out of the two plates

containing the expanding clones, one was used for the analysis of the expression of the

transgene using immunocytochemistry as described in the next section. From the other

plate genomic DNA was isolated from the cells and “Non-activation/Activation PCR was

performed on them as described below.

To check for the in-vitro cre recombination in ES cell clones, a PCR strategy was designed to

analyze cre recombination. Three different kinds of primers were designed to distinguish

between “- cre-recombined” and “+ cre-recombined clones. Primer a bind before the first

loxP site in the transgene construct (Fig. 3.12, A, blue arrow), primer b in the silencing

cassette (Fig. 3.12, A, orange arrow) and primer c after the second loxP site (Fig. 3.12, A, red

arrow). PCR which was carried out using primer pair a/b was called as “non-activation”

PCR and that with primer pair a/c as “activation” PCR.

For the non-activation PCR, a product size of 226 bp is expected with the primer pair a/b∼

(representative Fig. 3.12, A). For the activation PCR, a product of 5157 bp and 186 bp is∼

expected with primer pair a/c depending upon the clones (representative Fig. 3.12, A).

Page | 84

RESULTS

Non-activation PCR was carried out on “- cre-recombined” and “+ cre-recombined” clones.

PCR product of 226 bp was obtained in all “- cre recombined” clones (Fig. 3.12, B, upper∼

gel). A similar band pattern was also observed for the “+ cre recombined” clones (Fig. 3.12,

B, lower gel). This can be explained with the fact that not all the expanding colonies express

cre recombinase but still expressed the silenced transgene construct.

Similarly, activation PCR was also performed on “- cre-recombined” and “+ cre-recombined”

clones. The “- cre-recombined” clones did not show and product since the two primers bind

quite far away from each other and the region in between them could not be amplified (Fig.

3.12, B, upper gel). However, a band of 186 bp was observed for “+ cre recombined”∼

clones (Fig. 3.12, B, lower gel). This proved that cre-recombination occurred in the cells

which were electroporated with cre recombinase. The absence of any band in “- cre-

recombined” lanes shows that the band is specific for cells expressing cre recombinase. The

lack of specific product for clone 11 is an experimental artifact due to less quantity of DNA

used in the PCR.

Page | 85

RESULTS

Fig.3.12: PCR to check for the activation of the transgene in ES cells(A) Representative diagram of the transgene construct showing the primer binding sites and the product size expected. Primer a is depicted ad blue arrow, primer b as orange and primer c as red arrow. (B) Depicts the PCR results for Non-activation and Activation PCR using primer pairs a/b and a/c respectively. (*) stands for – cre recombined clones and (**) for + cre recombined clones. The positive control used in the PCR is the plasmid ilacZ/ I LacZ/GlyT1-EGFP for the non-activation PCR and I LacZ/GlyT1-EGFP for activationΔ Δ PCR.

Page | 86

RESULTS

3.1.6.2 X-Gal and DAB staining of the ES cell clones

To substantiate the results obtained from non-activation/activation PCR, X-Gal and DAB

staining was performed on - cre electroporated and + cre electroporated clones to confirm

the in-vitro cre recombination in the clones. For the X-Gal staining, the cells were treated as

described in section (2.2.8.9). DAB staining was performed as described in section (2.2.9.9).

Out of the four clones analyzed for the PCR, clone 12, clone 269, and clone 149 were chosen

for further analysis.

The cells untreated with cre recombinase (- cre recombinase) showed a homogeneous -galβ

expression (Fig. 3.13, A). However, upon expression of cre recombinase (+ cre

recombinase), there is a reduction in the number of cells expressing -gal, although someβ

cells expressing -gal can still be seen (Fig. 3.13, A). This can be explained by the fact thatβ

not all the cells electroporated expressed cre recombinase to allow the recombination to

occur. Hence, the transgene is still silenced in the cells. However, in cells which express cre

recombinase, the cre recombination leads to excision of the LacZ/Neor cassette, thereby not

expressing the -Gal.β

To check for the expression of mycGlyT1 and EGFP upon cre expression, DAB stain using

anti-myc and anti-EGFP antibody was performed. The EFFP expression in ES cells could not

be detected using fluorescence microscopy since these cells have high auto fluorescence.

Both the - cre electroporated and + cre electroporated clones were analyzed using anti

EGFP antibody. The - cre electroporated clones did not show any signal upon detection.

However, cells treated with cre recombinase, showed expression of EGFP as can be seen by

dark brown aggregates in the cell (Fig 3.13, B). This showed that there was an expression of

EGFP upon cre recombination. This also implied that the loxP sites in the transgene

construct were functional. Staining with anti-myc antibody was also tried. However, this

antibody was not sensitive enough to be used for this staining.

Together, the PCR data and the staining confirmed that the transgene construct could be

activated in vitro. Both of the clones were further used for blastocyst injection to generate

Page | 87

RESULTS

the transgenic mice.

Page | 88

Fig.3.13: in vitro cre activation of the transgene in ES cells(A) Shows the LacZ staining of the ES cell clones 12 and 149 before and after cre recombinase expression.

(B) DAB staining of the ES cell clones 12 and 149 before and after cre treatment. The cells were stained with anti-EGFP antibody to check for the expression of EGFP. The cells with the dark brown aggregates express EGFP upon cre expression. Scale bar: 50 µm

RESULTS

3.1.7 Production of the chimeric mice by the blastocyst injection of the ES cells

Production of chimeric mice by injecting embryonic stem cells into the cavity of a

mouse blastocyst is a critical step in the generation of mutant mouse models. Following

blastocyst injections, the ES cells become incorporated within the developing inner cell

mass of the embryo and contribute towards different embryonic lineages including the

germline cells which could then help in stable expression of the transgene over generations.

Chimeric mice resulting from the injected blastocysts are composed of tissue derived from

the inner cell mass of the host blastocyst as well as from the ES cells.

ES cell clones 12 and 149 were used for injecting mouse blastocysts for the generation of

the inGlyT1 transgenic mice. 3 days before the blastocyst injections, the ES cells from the

respective clones were thawed and expanded on 6-well plates. A representative picture of

the clones used before the injections is shown in Fig. 3.14, A. The injection of ES cells was

done by Frank Zimmermann from the Centre of Molecular Biology, University of

Heidelberg. In total, 18 blastocysts were injected with for both the clones. The

microinjected blastocysts were transplanted into pseudopregnant female mice. 5 founder

animals were obtained from both the clones (Fig. 3.14, B). For the ES cell clone 12, 2 of the

founders showed > 70% germline transmission. For the clone 149, 1 founder showed >

50% germline transmission. All these 3 founder animals were then mated out with C57/Bl6

animals to establish different inGlyT1 lines.

2 different inGlyT1 mouse lines were established, one from clone 12 (Line 12) and other

from clone 149 (Line 149).

Page | 89

RESULTS

Fig. 3.14: Production of the chimeras by the blastocyst injection of the ES cells(A) ES cell from clone 12 and 149 during expansion (upper panel) and before blastocyst injection (lower panel). (B) Chimeras obtained after injection.

3.1.7.1 Characterization of inGlyT1 transgenic mice

X-Gal staining of the tail biopsies and PCR were routinely preformed for the

characterization of the inGlyT1 transgenic mice

For the X-Gal staining, a piece of the tail was fixed in 0.2 % glutaraldehyde solution for 5

min. The tail was then washed twice with LacZ washing solution (see 2.1.11) and then

incubated in LacZ staining solution overnight at 37 °C.

Tail samples from animals from both the transgenic lines showed stained tails in contrast to

the wild type tail which did not express the transgene (Fig. 3.15, A). This confirmed that the

transgene was indeed expressing in the offsprings from the founder animals chosen for

breeding.

DNA was also extracted from mouse tails (see 2.2.2.4 and 2.2.2.5) and genotyping PCR was

performed on the samples using primer pair 44S/44AS. Both these primers bind within the

Page | 90

RESULTS

GlyT1 cDNA and upon amplification produce a product of 497 bp. As can be seen in Fig.∼

3.15, B, animal# 2, 5, and 6 show a band of 497 bp specific to GlyT1 upon amplification. In∼

contrast, animal # 1 and 3 does not show any band which conferred that these animals do

not express the transgene.

Fig. 3.15: X-Gal staining and genotyping on the mouse tails from inGlyT1 transgenic mice(A) X-Gal staining on the tail biopsies from the animals from inGlyT1 mice. (B) Representative gel showing PCR from the DNA samples isolated from tails of inGlyT1 mice.

Page | 91

RESULTS

3.1.7.2 -gal expression in β inGlyT1 transgenic mice

In order to check for the overall expression pattern of the transgene in the inGlyT1

animals, it was thought to perform whole mount embryo X-Gal staining. For this, E12.5

embryos were dissected from pregnant females and X-gal staining was performed as

described in Cold Spring Har. Protoc.: 2007. In short, 12.5 dpc embryos were dissected from

pregnant females and made free of their extraembryonic membranes in PBS. The embryos

were fixed in a fixing solution (see 2.1.11) at 4 °C for 30-60 min. Embryos were then

washed thrice with washing solution at room temperature for 20 min each and placed in

LacZ staining solution (see2.1.11) till they turned blue. The embryos were later washed

with PBS and serially desiccated in 50 %, 70 %, and 100 % ethanol before taking

photographs (protocol from Black lab, UCSF).

For the further analysis in the study, only the mouse Line #12 was analyzed since there

were some initial breeding problems with the Line #149.

As can be seen in Fig. 3.16, A, the wild-type embryos were colorless upon LacZ staining.

However, embryos expressing the transgene showed a homogeneous LacZ expression in

spinal cord, developing hindbrain, forebrain, eye, heart etc. This meant that the transgene

was ubiquitously expressed in all developing organ types. Fig. 3.16, B shows the magnified

images of the stained spinal cord, developing forebrain and developing hindbrain. In the

developing spinal cord the somites are also stained (Fig. 3.16, A). Stained telecephalic,

diencephalic and midbrain regions were also seen in developing forebrain and the stained

hindbrain (Fig. 3.16, B).

Page | 92

RESULTS

Fig. 3.16: Expression of the transgene in inGlyT1 transgenic mice(A) LacZ staining of 12.5 dpc embryos. The wild type embryo does not show any LacZ stain. However, the embryo expressing the transgene shows a homogeneous LacZ expression. Legends: fb-forebrain, mb-midbrain, ey-eye, ea-ear placode, np-nasal placode, hb-hindbrain, ht-heart, so-somites, fl-forelimbb, sc-spinal cord, hl-hindlimb. (B) Enlarged view of spinal cord, developing forebrain with different regions and developing hindbrain

Page | 93

RESULTS

3.1.7.3 Expression of -gal in the brain of β inGlyT1 transgenic mice

To check for the expression of the transgene in brain, LacZ staining was performed

on the brain sections (see2.2.8.7and 2.2.8.8) from the transgenic mice. The mice were first

identified as positives by LacZ staining of the tail and PCR and then their brain further

processed for the staining.

As depicted in Fig. 3.17, A, there was a homogeneous LacZ stain over all the regions of the

brain, showing that the transgene was ubiquitously expressed in all major regions.

However the level of expression in different regions varied. High expression was observed

in the hippocampus, cortex, and the olfactory bulb. Cerebellum on the other hand showed

much lower level of expression (Fig. 3.17, B). The different region of the hippocampus (CA1,

CA2, and CA3) could also be identified (Fig. 3.17, B).

Fig. 3.17: Expression pattern of -gal in brain of β inGlyT1 transgenic mice

Page | 94

RESULTS

(A) Depicts the LacZ staining on the whole brain section of inGlyT1 transgenic mice. (B) Shows the enlarged images of different stained regions of the brain: Hippocampus, Cortex, Cerebellum, and Olactory bulb. Abb: O: Stratum oriens; P: Stratum pyramidale; R: Stratum radiatum; LM: Stratum lucidum; M: Stratum molecular; G: Stratum granulosum; H: Hilus; DG: Dentate gyrus and CA: cornu ammonis.

3.1.7.4 Transgene expression upon expression of cre recombinase

After the mouse line was checked for expression of the transgene, it was investigated

whether the cre recombinase mediated excision of the LacZ/Neor silencing cassette could

lead to expression of the mycGlyT1 and EGFP upon recombination. For this, transgene

positive animals were mated with Synapsin cre mouse line, where the expression of cre

recombinase is under the control of neuron-specific Synapsin I promoter. Double transgenic

animals i.e animals positive for both GlyT1 and cre (Tg+/cre+) were used for analysis. As a

negative control, animals only positive for GlyT1 but negative for cre were used. For the

analysis, 3 animals from each litter set were used.

In the sections from the double transgenic mice, no EGFP fluorescence was detected upon

checking with fluorescence microscopy (data not shown). Imagining that the level of cre

expression varies from animal to animal and the rate of recombination being low, staining

was performed using anti-EGFP antibody to enhance the sensitivity.

In brain slices from (Tg+/cre+) mice, no EGFP immunreactivity was observed in any of the

brain regions analyzed (Fig. 3.18). The background staining was similar as compared to the

negative control samples. Stainings were also performed using anti-myc antibody.

However, no specific staining was observed in those animals as well (data not shown).

Different matings were also set up with other cre expressing lines such as GFAP cre (Glial

Fibrillary Acid Protein, Glia specific cre) and EmxI cre (forebrain specific cre). However, the

double transgenic animals from these matings too did not show any EGFP fluorescence

(data not shown).

Page | 95

RESULTS

Fig. 3.18: Immunostaining of the Tg+/Syn cre+ mice showing transgene activationImmunofluorescence in different regions of the brain from Tg+/Syn cre+ double transgenic mice. No EGFP-specific immunreactivity was observed in these mice. The staining is comparable to the negative control, where the transgene is non-activated. The cell nuclei are stained in blue with DAPI. Scale bar: 20 µm.

Page | 96

RESULTS

3.1.7.5 Transgene expression over generations

Since no activation of the transgene and EGFP fluorescence was observed upon

mating with different cre lines, different reasons were thought which could hamper with

the transgene activation. One reason was there might not be enough cre expression which

could lead to transgene activation. However, with the number of enough number of animals

and cre lines analyzed, this was ruled out. The other probability could be that the robust

expression of the transgene was lost upon generations. To verify this claim, transgenic mice

from four subsequent generations were analyzed for transgene expression by X-Gal

staining. Three sets of animals from different set of litters were analyzed per generation to

rule out any animal to animal variation.

As can be seen in Fig. 3.19, there was a subsequent loss of transgene expression over the

generation. The expression seemed to be robust during the N1 generation, but got

substantially reduced over subsequent generations. Different regions of the brain were

analyzed to also check for region to region variation of the transgene expression. However,

as can be seen in Fig. 3.19, there was loss of expression in most brain regions. There was so

substantial loss of the expression that the expression in the tail by X-Gal staining could also

not be detected reliably (data not shown).

Thus, it can be concluded from these findings that the expression of the transgene reduced

over the generation. Since the cells expressing the transgene reduced dramatically, the

probability of the cells expressing cre got further reduced. This even lowered the

probability expression of both cre and the transgene in the same cells. Thus, it was deduced

that with so less number of cells expressing the transgene in subsequent generation, the

probability of getting cells expressing both transgene and cre is minimal.

Page | 97

RESULTS

Fig. 3.19: Transgene expression over generationsRepresentative figure showing loss of transgene expression over generations. There was a substantial loss of expression of the transgene in most of the brain regions analyzed from generation N1 to N3.

Page | 98

RESULTS

RESULTS (PART II)

Page | 99

RESULTS

3.2 Developmental expression pattern of Glycine transporters

As described previously in this thesis, the neurotransmitter action of glycine is

terminated by the action of two glycine specific transporters: GlyT1 and GlyT2. These two

transporters act synergistically to remove the extracellular glycine from the synaptic cleft

(Eulenburg et. al., 2005). Knockout mice of both GlyT1 and GlyT2 were generated

previously to understand the physiological functions of these two transporters at the

synapses (Gomeza et. al., 2003 a and b). The phenotypes observed in these mice indicated

that early neonatal lethality in the GlyT1-/- mice is caused by the loss of glial GlyT1 in brain

stem and spinal cord. This contrasts with the early postnatal lethality of GlyT2-/- mice (P10-

P15) due to the loss of GlyT2 in the caudal regions of the brain.

Due to the contrasting phenotypes observed in the knockout mice for both the transporters,

it was suggested that the functions of these two transporters might differ in the mature CNS

from those at the neonatal stages. Earlier reports on the expression profiles of the two

transporters in mice showed that the mRNA for GlyT1 is detected as early as E 9 and E l0,

increased significantly at E 13 and remain at high levels up to E 15 (Adams et. al., 1995). In

contrast, the expression of GlyT2 was detected at E 11 which increased by E 15. Also, the

regions of the embryo expressing GlyT2 were quite distinct from those expressing GlyT1

(Adams et. al., 1995). The expression pattern of the two transporters over development also

varies amongst different species. However, no molecular data is available till date to clearly

define the expression pattern of these two transporters over different stages of

development. The second part of the study described in this thesis, discuss the expression

pattern and role of the two glycine transporters over development.

To check for the expression pattern of these two transporters over development,

membrane preparations were prepared from different aged C57/Bl6 mice. Glycine uptake

experiments and western blots were performed on the membrane preparations and the

expression pattern of these two transporters was analyzed.

Page | 100

RESULTS

3.2.1

Page | 101

RESULTS

Appendix I: List of oligonucleotides used in this study

Primer Oligosequence (5’-3’) Features

44S AGG CGT GGG CTA TGG TAT GAT G Genotyping GlyT1 Tg mice

44AS GAA CAA ACA GAA TGG TCA GCA CC Genotyping GlyT1 Tg mice

45S TGT GGA TGA GGT AGG GAA TGA GTG Genotyping GlyT1 Tg mice

45AS ACT GGT AGT GGT TGT AGG TGA TTG G Genotyping GlyT1 Tg mice

CS 26S ACC AGC CAG CTA CTA TCA ACT C Cre recombinase, genotyping

CS 26AS TAT ACG CGT GCT AGC GAA GAT CTC CAT

CTT CCA GCA G

Cre recombinase, genotyping

Cre a1 TAC AGC TCC TGG GCA ACG TG Non activation PCR, binds in the promoter region

Cre b1 AAC GCC AGG GTT TTC CCA Non activation PCR, binds in the LacZ region

Cre c4 CAA CAT CCC TTT GGC ACC Activation PCR, binds in the myc-GlyT1 start region

mcf ScaI TTC CTC CTC TCC TGA CTA CTC Multicopy PCR forward

mcb ScaI CGC ATA CAC TAT TCT CA GAA Multicopy PCR backward

YFP4 GAC CAC TAC CAG CAG AAC AC Sequencing EGFP

PolyA b2 CAG CTA TGA CTG GGA GTA G Sequencing poly A tail

LoxP F1

CS 1S GTC GGA TCC GCA CTG AAC GCA AGA GTC TG

Sequencing plasmid

CS 1AS CTC CTC GGA CAT CAG CTT CTG CTC CAA CAT CCC TTT GGC ACC

Sequencing plasmid

CS 2S CAG AAG CTG ATC TCC GAG GAG GAC CTG AAT GGT GCT GTC CCC AGC

Sequencing plasmid

CS 4AS CAG CTC GAG TCA GGA ACA CTG GTC ACG AG

Sequencing plasmid

All oligonucleotides were obtained from Metabion (Berlin, Deutschland).

Page | 102

RESULTS

Appendix II: Description of primer pairs used in this study and the size of

the amplified product obtained

Primer pair Purpose Annealing temp

Polymerase Product size

44S/44AS Genotyping inGlyT1 Tg mice

68 °C PAN scriptTM

polymerase497 bp∼

26S/26AS Genotyping Cre mice 56 °C Qiagen hotstart polymerase mix

300 bp∼

Cre a1/b1 Activation/Non-activation PCR

Qiagen hotstart polymerase mix

Cre a1/c4 Activation/Non-activation PCR

Qiagen hotstart polymerase mix

Mcf ScaI/Mcb ScaI

Multicopy PCR Qiagen hotstart polymerase mix

Page | 103

RESULTS

Page | 104

Combined thesis 1

Education

Transcript of Combined thesis 1