Einführung in die Biophysik - uni-muenchen.de · artiﬁcial chromosome (YAC). " A YAC vector...

SMRT!Erzeugen von Proteinen mit frei wählbaren Eigenschaften "(recombinant proteins)"Herstellen von transgenen Tieren und Pflanzen mit neuartigen Gendiagnostik (genotyping)!Somatische Gentherapie!Die Physik des DNA Schmelzen!

!

Gentechnik!

Einführung in die Biophysik!Schwerpunktstudium "Biophysik !

!

!20. Juni 2011!

Joachim Rädler!

DNA-Sequencing "by Sangers!

Zur Anzeige wird der QuickTime™ Dekompressor „TIFF (Unkomprimiert)“

benötigt.


benötigt.

Figure 2. An electropherogram of a finished sequencing reaction. As the fragments from the sequencing reaction are resolved via electrophoresis, a laser reads the fluorescence of each fragment (blue, green, red or yellow) and compiles the data into an image. Each colour, or fluorescence intensity, represents a different nucleotide (e.g. blue for C) and reveals where that nucleotide is in the sequence!

Single Molecule Techniques Boost Innovation in Human Genome Sequencing!

Year Cost Technology Who Machine runs Authors Coverage2001 $300,000,000 Sanger (ABI) reference 251 42001 $100,000,000 Sanger (ABI) reference 100,000 274 52007 $10,000,000 Sanger (ABI) Venter 100,000 31 72008 $2,000,000 Roche(454) Watson 234 27 72008 $1,000,000 Illumina Leukemia 98 48 332008 $500,000 Illumina Asian 35 77 362008 $250,000 Illumina African 40 196 302009 $30,000 Helicos Quake 2 4 10

Third generation sequencing: "Single Molecule Real Time (SMRT)"

!

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U U C U UC

Incorporation events

2006: 25 bp read length, ~ 10^6 templates, (Science 2008)

2008: 30 bp read length, ~10^8 templates, 90 megabases/hour

helicos –!biosciences!

DNA Oligo Synthesis!

DMT = Dimethoxytrityl!

käuflich bis ca 100 bB!

!"#$%&'()(*+,-.+*"#/($0$12+

Recombinant Proteins!

!Herstellen geeigneter DNA-Sequenzen!Kombination der DNA-Sequenz mit Hilfs"genen" (Promotor, ...)!Verpacken der DNA-Sequenz in einen Vektor !Einbringen des Vektors in den Organismus!Selektion der veränderten Organismen!Vervielfältigung der geklonten Organismen!Anregen der Expression der gewünschten Proteine!Gewinnen der gewünschten Produkte (Proteine)!

!

Einzelschritte:!

Restriction endonucleases!

The DNA nucleotide sequences recognized by four widely used restriction nucleases. As in the examples shown, such sequences are often six base pairs long and "palindromic" (that is, the nucleotide sequence is the same if the helix is turned by 180 degrees around the center of the short region of helix that is recognized). The enzymes cut the two strands of DNA at or near the recognition sequence. For some enzymes, such as Hpa I, the cleavage leaves blunt ends; for others, such as Eco RI, Hind III, and Pst I, the cleavage is staggered and creates cohesive ends. Restriction nucleases are obtained from various species of bacteria: Hpa I is from Hemophilus parainfluenzae, Eco RI is from Escherichia coli, Hind III is fromHemophilus influenzae, and Pst I is from Providencia stuartii. !

Recombinant DNA!

The formation of a recombinant DNA molecule. The cohesive ends produced by many kinds of restriction nucleases allow two DNA fragments to join by complementary base-pairing (see Figure 7-2). DNA fragments joined in this way can be covalently linked in a highly efficient reaction catalyzed by the enzyme DNA ligase. In this example a recombinant plasmid DNA molecule containing a chromosomal DNA insert is formed.!

Polymerase at work!

:MCB1201!

Tinnefeld / Gaub / SS2010!

BPE §3.3.3! 15!


benötigt.

!

Siehe auch "Patrik Cramer "Genzentrum!


BPE §3.3.3! 16!

The making of a yeast artificial chromosome (YAC). "A YAC vector allows the cloning of very large DNA molecules. TEL, CEN, and ORI are the telomere, centromere, and replication origin sequence elements, respectively, for the yeast Saccharomyces cerevisiae. BamH1 and EcoR1 are sites where the corresponding restriction nucleases cut the DNA double helix. The sequences denoted as A and B encode enzymes that serve as selectable markers to allow the easy isolation of yeast cells that have taken up the artificial chromosome. (Adapted from D.T. Burke, G.F. Carle, and M.V. Olson, Science 236:806-812, 1987. ¬© 1987 AAAS.)!

Mycoplasma laboratorium - the minimal genome project Craig Venter et al !

Start: Mycoplasma Genitalicum,

480 Genes , 265-350 Genes estimated to be essential for survival under lab conditions

Synthesis of the DNA completed January 2008!

381 gens consiting of 580.000 bps

Currently reconstruction of the chromosome

Future: Reduction gene by gene

The green fluorescent protein:"discovery, expression and development "!

Osamu Shimomura!Martin Chalfie!Roger Tsien!!

Nobel Prize in Chemistry 2008!

The chromophore!

Residues 65-67 (Ser-Tyr-Gly) in the GFP sequence spontaneously!form the fluorescent chromophore p-hydroxybenzylideneimidazolinone!!

The GFP family !and its mutants!

sample is extracted from the ampoule andcleaned of the surrounding excess impreg-nant by standard mechanical polishingtechniques. In this way, we preparednanowires of various metals (In, Sn, andAl) and semiconductors (Se, Te, GaSb,and Bi2Te3) (Fig. 2).

The nanowire composites create substan-tial electric field patterns over the samplesurface. We used a scanning probe microscopeto measure electric fields at the surface of ananocomposite. In a NanoScope (Digital In-struments, Santa Barbara, California) scan-ning force microscope, the sample is mountedwith conductive epoxy to a metal holder andis held at a few volts relative to a conductivecantilever tip that is grounded. The metal-coated, etched, single-crystal silicon tip has aradius of curvature of about 5 nm. The tip isset to oscillate at a frequency near its reso-nance frequency (78 kHz). When the canti-lever encounters a vertical electric field gradi-ent, the effective spring constant is modified,shifting its resonance frequency. By recordingthe amplitude of the cantilever oscillationswhile scanning the sample surface, we obtainan image that reveals the strength of theelectric force gradient (13, 14).

The image, however, may also containtopographical information; it is difficult toseparate the two effects. This is circumvent-ed by taking measurements in two passesover each scan line (15). On the first pass,a topographical image (Fig. 3A) is takenwith the cantilever tapping the surface, andthe information is stored in memory. Onthe second pass, the tip is lifted to aselected separation between the tip andlocal surface topography (typically 20 to200 nm), such that the tip does not touchthe surface. By using the stored topograph-ical data instead of the standard feedback,we can keep the separation constant. Inthis second pass, cantilever oscillation am-plitudes are sensitive to electric force gradi-ents without being influenced by topo-graphic features (Fig. 3B). This two-passmeasurement process is recorded for everyscan line, producing separate topographicand electric force images. From these imag-es, contours of electric force gradient (Fig.3C) can be drawn.

The amplitude of the cantilever oscilla-tions is very large for small lift heights, andthe images fade at separations larger than 80nm. This is consistent with previous reports ofa strong dependence of the tip-surface forceon the vertical separation (13). More workneeds to be done to understand this quantita-tively. Note that some of the nanowires thatappear in the topographic image are missingfrom the electric field image (Fig. 3). This isbecause either electrical contact to thesenanowires has failed or electrical conductionalong the wire length has been interrupted.The scanning force technique thus provides a

802

unique way of mapping the electrical proper-ties of nanocomposites.

Applications of the metal nanowire com-posites include high-density electrical multi-feedthroughs and high-resolution plates fortransferring a two-dimensional charge distri-bution between microelectronic devices. Thesemiconductor nanowires can be used in pho-todetector arrays of high spatial resolution,where each wire acts as a pixel ofsubmicrome-ter dimensions. Also, with the application ofthe injection technique to ultrasmall channelinsulators (channel diameter less than 50 nm)(16, 17), nanowire arrays can be made forfundamental studies of a variety of phenome-na, such as quantum confinement of chargecarriers and mesoscopic transport.

REFERENCES AND NOTES1. An overview can be found in M. J. Yacaman, T.

Tsakalakos, B. H. Kear, Eds., Nanostruct. Mat. 3(nos. 1-6) (1993).

2. R. Roy, Nanophase and Nanocomposite Materi-als, S. Komarneni, J. C. Parker, G. J. Thomas,Eds. (Mat. Res. Soc. Symp. Proc. 286, MaterialsResearch Society, Pittsburgh, PA, 1993), pp.241-250, and references therein.

3. Early work on the preparation of ultrathin super-conducting wires by injection of nanochannelmatrices was done by W. G. Schmidt and R. J.Charles [J. Appl. Phys. 35,2552 (1964)] and byV.N. Bogomolov [Sov. Phys. Usp. 21, 77 (1978)].

4. C. A. Huber and T. E. Huber, J. Appl. Phys. 64,6588 (1988).

5. N. F. Borrelli and J. C. Luong, Proc. SPIE866, 104(1988).

6. B. L. Justus, R. J. Tonucci, A. D. Berry, Appl.Phys. Lett. 61, 3151 (1992).

7. M. J. Tierney and C. R. Martin, J. Phys. Chem. 93,2878 (1989).

8. G. D. Stucky and J. E. Mac Dougall, Science 247,669 (1990), and references therein.

9. P. M. Ajayan and S. lijima, Nature361,333 (1993).10. An array of parallel metal cylinders would be

transparent to light that had a wavelength muchlarger than the cylinder diameter and separationand propagated along the cylinder axis [D. E.Aspnes, A. Heller, J. D. Porter, J. Appl. Phys. 60,3028 (1986)].

11. Whatman Laboratory Division, Clifton, NJ.12. J. W. Diggle, T. C. Downie, C. W. Goulding, J.

Electrochem. Soc. 69, 365 (1969).13. Y. Martin, D. W. Abraham, H. K. Wickramasinghe,

Appl. Phys. Lett. 52, 1103 (1988).14. J. E. Stern, B. D. Terris, H. J. Maimin, D. Rugar,

ibid. 53, 2717 (1988).15. Lift Mode operation, Digital Instruments, Inc., San-

ta Barbara, CA (patent pending).16. R. C. Furneaux, W. R. Rigby, A. P. Davidson,

Nature 337, 147 (1989).17. R. J. Tonucci, B. L. Justus, A. J. Campillo, C. E.

Ford, Science 258, 783 (1992).18. We thank V. Elings for valuable discussions, B.

Schardt and S. Thedford for image processing,and S. Nourbakhsh for electron microscopy. Thiswork was supported by the Army Research Office,the Independent Research Program of the Officeof Naval Research, and the National ScienceFoundation.

22 October 1993; accepted 20 December 1993

Green Fluorescent Protein as aMarker for Gene Expression

Martin Chalfie,* Yuan Tu, Ghia Euskirchen, William W. Ward,Douglas C. Prasherf

A complementary DNAfor the Aequorea victoria green fluorescent protein (GFP) producesa fluorescent product when expressed in prokaryotic (Escherichia coli) or eukaryotic(Caenorhabditis elegans) cells. Because exogenous substrates and cofactors are notrequired for this fluorescence, GFP expression can be used to monitor gene expressionand protein localization in living organisms.

Light is produced by the bioluminescentjellyfish Aequorea victoria when calciumbinds to the photoprotein aequorin (1).Although activation of aequorin in vitro orin heterologous cells produces blue light,the jellyfish produces green light. This lightis the result of a second protein in A.victoria that derives its excitation energyM. Chalfie, Y. Tu, G. Euskirchen, Department of Bio-logical Sciences, Columbia University, New York, NY10027, USA.W. W. Ward, Department of Biochemistry and Micro-biology, Cook College, Rutgers University, New Bruns-wick, NJ 08903, USA.D. C. Prasher, Biology Department, Woods HoleOceanographic Institution, Woods Hole, MA 02543,USA.*To whom correspondence should be addressed.fPresent address: U.S. Department of Agriculture,Building 1398, Otis Air National Guard Base, MA02542, USA.

SCIENCE * VOL. 263 * 11 FEBRUARY 1994

from aequorin (2), the green fluorescentprotein (GFP).

Purified GFP, a protein of 238 aminoacids (3), absorbs blue light (maximally at395 nm with a minor peak at 470 nm) andemits green light (peak emission at 509 nmwith a shoulder at 540 nm) (2, 4). Thisfluorescence is very stable, and virtually nophotobleaching is observed (5). Althoughthe intact protein is needed for fluores-cence, the same absorption spectral proper-ties found in the denatured protein arefound in a hexapeptide that starts at aminoacid 64 (6, 7). The GFP chromophore isderived from the primary amino acid se-quence through the cyclization of serine-dehydrotyrosine-glycine within this hexa-peptide (7). The mechanisms that producethe dehydrotyrosine and cyclize the poly-

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SCIENCE * VOL. 263 * 11FEBRUARY 1994!

sample is extracted from the ampoule andcleaned of the surrounding excess impreg-nant by standard mechanical polishingtechniques. In this way, we preparednanowires of various metals (In, Sn, andAl) and semiconductors (Se, Te, GaSb,and Bi2Te3) (Fig. 2).

The nanowire composites create substan-tial electric field patterns over the samplesurface. We used a scanning probe microscopeto measure electric fields at the surface of ananocomposite. In a NanoScope (Digital In-struments, Santa Barbara, California) scan-ning force microscope, the sample is mountedwith conductive epoxy to a metal holder andis held at a few volts relative to a conductivecantilever tip that is grounded. The metal-coated, etched, single-crystal silicon tip has aradius of curvature of about 5 nm. The tip isset to oscillate at a frequency near its reso-nance frequency (78 kHz). When the canti-lever encounters a vertical electric field gradi-ent, the effective spring constant is modified,shifting its resonance frequency. By recordingthe amplitude of the cantilever oscillationswhile scanning the sample surface, we obtainan image that reveals the strength of theelectric force gradient (13, 14).

The image, however, may also containtopographical information; it is difficult toseparate the two effects. This is circumvent-ed by taking measurements in two passesover each scan line (15). On the first pass,a topographical image (Fig. 3A) is takenwith the cantilever tapping the surface, andthe information is stored in memory. Onthe second pass, the tip is lifted to aselected separation between the tip andlocal surface topography (typically 20 to200 nm), such that the tip does not touchthe surface. By using the stored topograph-ical data instead of the standard feedback,we can keep the separation constant. Inthis second pass, cantilever oscillation am-plitudes are sensitive to electric force gradi-ents without being influenced by topo-graphic features (Fig. 3B). This two-passmeasurement process is recorded for everyscan line, producing separate topographicand electric force images. From these imag-es, contours of electric force gradient (Fig.3C) can be drawn.

The amplitude of the cantilever oscilla-tions is very large for small lift heights, andthe images fade at separations larger than 80nm. This is consistent with previous reports ofa strong dependence of the tip-surface forceon the vertical separation (13). More workneeds to be done to understand this quantita-tively. Note that some of the nanowires thatappear in the topographic image are missingfrom the electric field image (Fig. 3). This isbecause either electrical contact to thesenanowires has failed or electrical conductionalong the wire length has been interrupted.The scanning force technique thus provides a

802

unique way of mapping the electrical proper-ties of nanocomposites.

Applications of the metal nanowire com-posites include high-density electrical multi-feedthroughs and high-resolution plates fortransferring a two-dimensional charge distri-bution between microelectronic devices. Thesemiconductor nanowires can be used in pho-todetector arrays of high spatial resolution,where each wire acts as a pixel ofsubmicrome-ter dimensions. Also, with the application ofthe injection technique to ultrasmall channelinsulators (channel diameter less than 50 nm)(16, 17), nanowire arrays can be made forfundamental studies of a variety of phenome-na, such as quantum confinement of chargecarriers and mesoscopic transport.

REFERENCES AND NOTES1. An overview can be found in M. J. Yacaman, T.

Tsakalakos, B. H. Kear, Eds., Nanostruct. Mat. 3(nos. 1-6) (1993).

2. R. Roy, Nanophase and Nanocomposite Materi-als, S. Komarneni, J. C. Parker, G. J. Thomas,Eds. (Mat. Res. Soc. Symp. Proc. 286, MaterialsResearch Society, Pittsburgh, PA, 1993), pp.241-250, and references therein.

3. Early work on the preparation of ultrathin super-conducting wires by injection of nanochannelmatrices was done by W. G. Schmidt and R. J.Charles [J. Appl. Phys. 35,2552 (1964)] and byV.N. Bogomolov [Sov. Phys. Usp. 21, 77 (1978)].

4. C. A. Huber and T. E. Huber, J. Appl. Phys. 64,6588 (1988).

5. N. F. Borrelli and J. C. Luong, Proc. SPIE866, 104(1988).

6. B. L. Justus, R. J. Tonucci, A. D. Berry, Appl.Phys. Lett. 61, 3151 (1992).

7. M. J. Tierney and C. R. Martin, J. Phys. Chem. 93,2878 (1989).

8. G. D. Stucky and J. E. Mac Dougall, Science 247,669 (1990), and references therein.

9. P. M. Ajayan and S. lijima, Nature361,333 (1993).10. An array of parallel metal cylinders would be

transparent to light that had a wavelength muchlarger than the cylinder diameter and separationand propagated along the cylinder axis [D. E.Aspnes, A. Heller, J. D. Porter, J. Appl. Phys. 60,3028 (1986)].

11. Whatman Laboratory Division, Clifton, NJ.12. J. W. Diggle, T. C. Downie, C. W. Goulding, J.

Electrochem. Soc. 69, 365 (1969).13. Y. Martin, D. W. Abraham, H. K. Wickramasinghe,

Appl. Phys. Lett. 52, 1103 (1988).14. J. E. Stern, B. D. Terris, H. J. Maimin, D. Rugar,

ibid. 53, 2717 (1988).15. Lift Mode operation, Digital Instruments, Inc., San-

ta Barbara, CA (patent pending).16. R. C. Furneaux, W. R. Rigby, A. P. Davidson,

Nature 337, 147 (1989).17. R. J. Tonucci, B. L. Justus, A. J. Campillo, C. E.

Ford, Science 258, 783 (1992).18. We thank V. Elings for valuable discussions, B.

Schardt and S. Thedford for image processing,and S. Nourbakhsh for electron microscopy. Thiswork was supported by the Army Research Office,the Independent Research Program of the Officeof Naval Research, and the National ScienceFoundation.

22 October 1993; accepted 20 December 1993

Green Fluorescent Protein as aMarker for Gene Expression

Martin Chalfie,* Yuan Tu, Ghia Euskirchen, William W. Ward,Douglas C. Prasherf

A complementary DNAfor the Aequorea victoria green fluorescent protein (GFP) producesa fluorescent product when expressed in prokaryotic (Escherichia coli) or eukaryotic(Caenorhabditis elegans) cells. Because exogenous substrates and cofactors are notrequired for this fluorescence, GFP expression can be used to monitor gene expressionand protein localization in living organisms.

Light is produced by the bioluminescentjellyfish Aequorea victoria when calciumbinds to the photoprotein aequorin (1).Although activation of aequorin in vitro orin heterologous cells produces blue light,the jellyfish produces green light. This lightis the result of a second protein in A.victoria that derives its excitation energyM. Chalfie, Y. Tu, G. Euskirchen, Department of Bio-logical Sciences, Columbia University, New York, NY10027, USA.W. W. Ward, Department of Biochemistry and Micro-biology, Cook College, Rutgers University, New Bruns-wick, NJ 08903, USA.D. C. Prasher, Biology Department, Woods HoleOceanographic Institution, Woods Hole, MA 02543,USA.*To whom correspondence should be addressed.fPresent address: U.S. Department of Agriculture,Building 1398, Otis Air National Guard Base, MA02542, USA.


from aequorin (2), the green fluorescentprotein (GFP).

Purified GFP, a protein of 238 aminoacids (3), absorbs blue light (maximally at395 nm with a minor peak at 470 nm) andemits green light (peak emission at 509 nmwith a shoulder at 540 nm) (2, 4). Thisfluorescence is very stable, and virtually nophotobleaching is observed (5). Althoughthe intact protein is needed for fluores-cence, the same absorption spectral proper-ties found in the denatured protein arefound in a hexapeptide that starts at aminoacid 64 (6, 7). The GFP chromophore isderived from the primary amino acid se-quence through the cyclization of serine-dehydrotyrosine-glycine within this hexa-peptide (7). The mechanisms that producethe dehydrotyrosine and cyclize the poly-

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Fig. 1. Expression of GFP in E. coli. The bacte-ria on the right side of the figure have the GFPexpression plasmid. Cells were photographedduring irradiation with a hand-held long-waveUV source.

peptide to form the chromophore are un-known. To determine whether additionalfactors from A. victoria were needed for theproduction of the fluorescent protein, wetested GFP fluorescence in heterologoussystems. Here, we show that GFP expressedin prokaryotic and eukaryotic cells is capa-ble of producing a strong green fluorescencewhen excited by blue light. Because thisfluorescence requires no additional geneproducts from A. victoria, chromophore for-mation is not species-specific and occurseither through the use of ubiquitous cellularcomponents or by autocatalysis.

Expression of GFP in Escherichia coli (8)under the control of the T7 promoterresults in a readily detected green fluores-cence (9) that is not observed in controlbacteria. Upon illumination with a long-wave ultraviolet (UV) source, fluorescentbacteria were detected on plates that con-tained the inducer isopropyl-P-D-thioga-lactoside (IPTG) (Fig. 1). Because the

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Wavelength (nm)Fig. 2. Excitation and emission spectra of E.coli-generated GFP (solid lines) and purified A.victoria L form GFP (dotted lines).

cells grew well in the continual presenceof the inducer, GFP did not appear tohave a toxic effect on the cells. WhenGFP was partially purified from this strain(10), it was found to have fluorescenceexcitation and emission spectra indistin-guishable from those of the purified nativeprotein (Fig. 2). The spectral properties ofthe recombinant GFP suggest that thechromophore can form in the absence ofother A. victoria products.

Transformation of the nematode Cae-norhabditis elegans also resulted in the pro-duction of fluorescent GFP (II) (Fig. 3).GFP was expressed in a small number ofneurons under the control of a promoterfor the mec-7 gene. The mec-7 gene en-codes a P-tubulin (12) that is abundant insix touch receptor neurons in C. elegansand less abundant in a few other neurons(13, 14). The pattern of expression ofGFP was similar to that detected byMEC-7 antibody or from mec-7-lacZ fu-sions (13-15). The strongest fluorescencewas seen in the cell bodies of the fourembryonically derived touch receptor neu-rons (ALML, ALMR, PLML, and PLMR)in younger larvae. The processes fromthese cells, including their terminalbranches, were often visible in larval ani-mals. In some newly hatched animals, thePLM processes were short and ended inwhat appeared to be prominent growthcones. In older larvae, the cell bodies ofthe remaining touch cells (AVM andPVM) were also seen; the processes ofthese cells were more difficult to detect.These postembryonically derived cellsarise during the first of the four larvalstages (16), but their outgrowth occurs inthe following larval stages (17), with thecells becoming functional during thefourth larval stage (18). The fluorescenceof GFP in these cells is consistent withthese previous results: no fluorescence wasdetected in these cells in newly hatched orlate first-stage larvae, but fluorescence wasseen in four of ten late second-stage lar-

Fig. 3. Expression of GFP in afirst-stage C. elegans larva. Twotouch receptor neurons (ALMRand PLMR) are labeled at theirstrongly fluorescing cell bodies.Processes can be seen project-ing from both of these cell bodies.Halos produced from the out-of-focus homologs of these cells onthe other side of the animal areindicated by arrowheads. Thethick arrow points to the nervering branch from the ALMR cell(out of focus); thin arrows point toweakly fluorescing cell bodies.The background fluorescence isthe result of the animal's autofluo-rescence.

vae, all nine early fourth-stage larvae, andseven of eight young adults (19). In addi-tion, moderate to weak fluorescence wasseen in a few other neurons (Fig. 3) (20).

Like the native protein, GFP expressedin both E. coli and C. elegans is quite stable(lasting at least 10 min) when illuminatedwith 450- to 490-nm light. Some photo-bleaching occurs, however, when the cellsare illuminated with 340- to 390-nm or395- to 440-nm light (21).

Several methods are available to moni-tor gene activity and protein distributionwithin cells. These include the formation offusion proteins with coding sequences forP3-galactosidase, firefly luciferase, and bac-terial luciferase (22). Because such methodsrequire exogenously added substrates or co-factors, they are of limited use with livingtissue. Because the detection of intracellu-lar GFP requires only irradiation by nearUV or blue light, it is not limited by theavailability of substrates. Thus, it shouldprovide an excellent means for monitoringgene expression and protein localization inliving cells (23, 24). Because it does notappear to interfere with cell growth andfunction, GFP should also be a convenientindicator of transformation and one thatcould allow cells to be separated with fluo-rescence-activated cell sorting. We alsoenvision that GFP can be used as a vitalmarker so that cell growth (for example,the elaboration of neuronal processes) andmovement can be followed in situ, especial-ly in animals that are essentially transparentlike C. elegans and zebra fish. The relativelysmall size of the protein may facilitate itsdiffusion throughout the cytoplasm of ex-tensively branched cells like neurons andglia. Because the GFP fluorescence persistsafter treatment with formaldehyde (9),fixed preparations can also be examined. Inaddition, absorption of appropriate laserlight by GFP-expressing cells (as has beendone for Lucifer Yellow-containing cells)(25) could result in the selective killing ofthe cells.


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Single Cell GFP Expression

Transgenic species!

Transgenic species!

MCB0801!

Welt am Sonntag 14.1.08!

Okabe et al. FEBS Vol.407 p. 313 Science 1997 July 4; 277: 41

Transgene Mäuse und Schweine

Erzeugen von Proteinen mit frei wählbaren Eigenschaften über "Bakterien und andere Lebewesen (recombinat proteins)"!Herstellen von transgenen Tieren und Pflanzen mit neuartigen Eigenschaften (transgenic plants & animals)"!Gendiagnostik (genotyping)!!Patientenspezifische Pharmaentwicklung (pharmacogenomics)"!Somatische Gentherapie!

!

Gentechnik!!Ziele:!

Wirkstofftest mit DNA-Chips!(Expression profiling)!!Fragestellung: Schädigt ein neuer Wirkstoff die Leber?!!Vorgehen: Vergleich der Gene, die von dem neuen Wirkstoff in der Leberzelle aktiviert werden mit den von bekannten leberschädigenden Wirkstoffen aktivierten. (-> Expression Profiling)!!Technik: Chip, der mit verschiedenen einzelsträngigen DNA-Molekülen Schachbrettartig belegt ist (Mikroarray)!

Protokoll:"!1.) Behandle Leberzellen mit Wirkstoff; sammle m-RNA von diesen und unbehandelten Leberzellen; Hinweis: die Zellen erzeugen vornehmlich die m-RNA die aufgrund der Wirkstoffzugabe neu benötigt werden.!

!!2.) Baue zu beiden m-RNA-Typen komplementäre c-DNA auf (DNA ist stabiler !als RNA) die mit unterschiedlichen Fluoreszenzfarbstoffen markiert sind. Dazu und für !vieles andere wird die Polymerase Kettenreaktion (PCR) (3EPCR.mov) benutzt.!

3.) Die c-DNA wird auf den DNA-Chip gegeben und hybridisiert dort mit den jeweiligen komplementären Strängen des Chips !

4.) Mit einem Scanner werden die Fluoreszenzsignale der jeweiligen Punkte (Bindungsmuster) auf dem Chip gemessen. Damit erhält man für den neuen Wirkstoff ein Reaktionsmuster.!

!

5.) Das Aktivitätsmuster wird mit dem bekannter Wirkstoffe verglichen:"!

MGA1201A!

How to “ program“ a DNA Chip!

MGA1201B!

Functional genomics!

Where will we go?!

Smaller!

Faster!

Cheaper!

==> Single Molecule Assays!

==> Nano-Biotechnology!

Erzeugen von Proteinen mit frei wählbaren Eigenschaften über "Bakterien und andere Lebewesen (recombinat proteins)"!Herstellen von transgenen Tieren und Pflanzen mit neuartigen Eigenschaften (transgenic plants & animals)"!Gendiagnostik (genotyping)!!Patientenspezifische Pharmaentwicklung (pharmacogenomics)"!Somatische Gentherapie!

!

Gentechnik!!Ziele:!

DNA

cationicliposomes

nucleus

Golgi

ER

DNase

complex formation

endocytosis

endosomal breakup

nuclear translocation

k1

k2

k3

Gene Delivery Mediated by Synthetic Reagents - Transfer across many barriers

k0 Siehe !

Rädler, !

Bräuchle, !

Wagner!

nan

SFB

486

Self-Assembly bulk structure size distribution / stability single molecule assembly

Transport

The Physics of Nano-Medicine

Unpacking

in semidilute networks across membranes

gene expression

electroporation

non-viral chemical vectors

Ca++ phosphate cationic polymers cationic liposomes

microinjection mechanical methods

viral vectors retroviruses (Rv) Adenovirus (Ad) AAV, SV40

Gene transfer to eucaryotic cells


BPE §3.3.4! 42!

DNA /polycation NPs with virus-like functions!

++++

Targeting domains (select desired interaction)

Compacting domain

Shielding domains(block undesired interactions)

Transport domains(delivery across barriers)

Environment-specific activation/disassembly

(pH -, ionic-, redox- sensitive)

*

DNAcond.

* *

* *

**** *

**

**

**

*

*

****

++++


Compacting domain


++++


Compacting domain



Compacting domain





*




*

DNAcond.DNAcond.

* *

* *

**** *

**

**

**

*

*

****

Tf, EGF, sugars,antibodies

PEI, cleavable oligocations PEG

Amphipathic peptides, NLS

Hydrazone, succinate, disulfide linkage, peptides

Barriers for intravenous gene delivery

W. Li & F.C. Szoka, Pharm Res. vol. 24 (3) (2007)

.345#')0+6'378"8+!+9$+9)31"*":+;"("+<=>3"88'$(+

?@A  B(+C'C$+*)31"4(1D+E3$%+&0$$:+#'3#70)4$(+FG+*$+:'8*)(*+*)31"*+#"00+

+++++

" +873C'C)0+'(+&'$0$1'#)0+H7':8+" +>)88'(1+0'C"3I+8>0""(+" +"=*3)#"0070)3+%)*3'=+

?JA+K"0070)3+&)33'"38D+++++++FG+7>*)L"+'(*$+*)31"*+#"00+FG+*$+(7#0"78+

#"00+#70*73"+828*"%

'#+'(+C'C$++

" +"(:$#2*$8'8+" +3"0")8"+E3$%+"(:$8$%"8+" +#2*$>0)8%'#+*3)M#L'(1+" +(7#0")3+7>*)L"+

!"#$%&#'()&**+"*)+@N+>$$3+)##"88+*$+*)31"*+#"00+JN+0$O+*3)(8E"#4$(+"M#'"(#2+PN+0'%'*":+*3)(81"("+8'Q"+RN+'():"S7)*"+1"("+#$(*3$0+TN+>$$3+*)31"4(1+UN+'%%7("+3"8>$(8"+VN+%)(7E)#*73'(1+)(:+8)E"*2+'887"8+

47!

Passive tissue targeting is

achieved by extravasation of nanoparticles through increased

permeability of the tumour vasculature and ineffective

lymphatic drainage

!

47

EPR effect : #

Einführung in die Biophysik - uni-muenchen.de · artiﬁcial chromosome (YAC). " A YAC vector...

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Transcript of Einführung in die Biophysik - uni-muenchen.de · artiﬁcial chromosome (YAC). " A YAC vector...