Lineage Analysis Using Retrovirus Vectors--生物秀

A nested PCR was performed as follows. To each well was added 20 m l of the following reaction mix:

2 ml 10 X PCR buffer with Mg (Boehringer Mannheim)

0.15 ml BOLAPO 5 and 6 (0.6 mg/ml)

O.l5 ml dNTP mixture (25 mM) (Boehringer Mannheim)

17.4 ml water

0.4 ml Taq DNA polymerase (Boehringer Mannheim)

The reactions were cycled as follows:

93°C for 2.5 min, [94°C for 45 sec, 67°C for 2 min, 72°C for 2 min] X 33, 72°C for 5 min.

One ml of the above reaction was added to 20 ml of the same reaction mix, substituting BOLAPO 7 and 8 for BOLAPO 5 and 6. The amplification program was:

93°C for 2.5 min., [94°C for 45 sec, 72°C for 2 min] X 30, 72°C for 5 min.

Eight ml of the second reaction mix was added to 2.5 ml of gel loading buffer and then fractionated on a 3% NuSieve GTG/1% SeaKem ME agarose gel. Amplifications which yielded a DNA product of the appropriate molecular weight (bp) were sequenced using the Exo- Pfu Cyclist kit (Stratagene). One ml of nested PCR product was added to the following reaction mix:

0.15 ml BOLAPO 7 (0.6 mg/ml)

3 ml 10 X Sequencing buffer

0.75 ml 35S-(alpha)dATP

4 ml DMSO

12.1 ml water

l ml Exo- Pfu DNA polymerase

This reaction was mixed and 5 ml portions were added separately to 5 ml of the four dNTP/ddNTP mixtures. The reactions were overlayed with oil and cycled as follows:

95°C for 5 min [95°C for 30 sec, 60°C for 30 sec, 72°C for 1 min] X 30

The reactions were terminated by the addition of 5 ml of stop buffer, and then fractionated by 6% acrylamide gel electrophoresis.

At this point, 98 individual clones have been sequenced in our laboratory. All carry a unique DPLl insert. Of those, the degenerate oligo region was shorter than 24 bases in seven clones and longer in 1 clone. Occasional variations in the (GC)(AT) sequence were also seen.

SEQUENCES OF OLIGONUCLEOTIDES REFERENCED ABOVE

DPL2 - 5'-TAGGAGGCGCGCCTTT-[(GC)(AT)]12

GTTCTCGAGGACACCTGACTGGCTGAGGG

CTTCCGCGACCCGAGATCTCAGCTTCC-3

DPLl - 5'-TAGGAGGCGCGCCTTT-[(GC)(AT)]12

GTTACGCGTTAATTAACTCGAGATCTCAGCTTC-3'

DPLP- 5'-GAAGCTGAGATCTCGAGTTA-3'

DPLP5 - 5'-TAGGAGGCGCGCCTTT-3'

BOLAPO 5 - 5'-CCAGGGACTGCAGGTTGTGCCCTGT-3'

BOLAPO 6 - 5'-AGACACACATTCCACAGGGTCGAAG-3'

BOLAPO7-5'-GGCTGCCTGCACCCCAGGAAAGGAG-3'

BOLAPO8-5'-GGTCTCGGAAGCCCTCAGCCCAGTC-3' REFERENCES

Coffin, J.M., S.H. Hughes and H.E. Varmus, Retroviruses. 1997, Plainview, NY: Cold Spring Harbor Laboratory Press.
Roe, T.Y., T.C. Reynolds, G. Yu and P.O. Brown, EMBO J., 1993. 12: p. 2099-2108.
Naldini, L., U. Blomer, P. Gallay, D. Ory, R. Mulligan, F.H. Gage, I.M. Verma and D. Trono, Science, 1996. 272(5259): p. 263-7.
Ausubel, F.M., R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith and K. Struhl, Current Protocols in Molecular Biology. 1997, Arial, NY: Greene Publishing Associates.
Mann, R., R.C. Mulligan and D. Baltimore, Cell, 1983. 33(1): p. 153-9.
Danos, O. and R.C. Mulligan, Proc Natl Acad Sci U S A, 1988. 85(17): p. 6460-4.
Markowitz, D., S. Goff and A. Bank, J Virol, 1988. 62(4): p. 1120-4.
Morgenstern, J.P. and H. Land, Nucleic Acids Res, 1990. 18(12): p. 3587-96.
Pear, W.S., G.P. Nolan, M.L. Scott and D. Baltimore, Proc Natl Acad Sci U S A, 1993. 90(18): p. 8392-6.
Soneoka, Y., P.M. Cannon, E.E. Ramsdale, J.C. Griffiths, G. Romano, S.M. Kingsman and A.J. Kingsman, Nucleic Acids Res, 1995. 23(4): p. 628-33.
Yee, J.K., T. Friedmann and J.C. Burns, Methods Cell Biol, 1994. 43(Pt A): p. 99-112.
Boerkoel, C.F., M.J. Federspiel, D.W. Salter, W. Payne, L.B. Crittenden, H.J. Kung and S.H. Hughes, Virology, 1993. 195(2): p. 669-79.
Cosset, F.L., C. Legras, Y. Chebloune, P. Savatier, P. Thoraval, J.L. Thomas, J. Samarut, V.M. Nigon and G. Verdier, J Virol, 1990. 64(3): p. 1070-8.
Stoker, A.W. and M.J. Bissell, J Virol, 1988. 62(3): p. 1008-15.
Chen, C.-M.A., D.M. Smith, M.A. Peters, M.E.S. Samson, J. Zitz, C.J. Tabin and C.L. Cepko, Dev. Biol., 1999. 214: p. 370-384.
Schlegel, R., T.S. Tralka, M.C. Willingham and I. Pastan, Cell, 1983. 32(2): p. 639-46.
Gaiano, N., J.D. Kohtz, D.H. Turnbull and G. Fishell, Nat Neurosci, 1999. 2(9): p. 812-9.
Price, J., D. Turner and C. Cepko, Proc Natl Acad Sci U S A, 1987. 84(1): p. 156-60.
Galileo, D.S., G.E. Gray, G.C. Owens, J. Majors and J.R. Sanes, Proc Natl Acad Sci U S A, 1990. 87(1): p. 458-62.
Fields-Berry, S.C., A.L. Halliday and C.L. Cepko, Proc Natl Acad Sci U S A, 1992. 89(2): p. 693-7.
Fekete, D.M., J. Perez-Miguelsanz, E.F. Ryder and C.L. Cepko, Dev Biol, 1994. 166(2): p. 666-82.
Chalfie, M., Y. Tu, G. Euskirchen, W.W. Ward and D.C. Prasher, Science, 1994. 263(5148): p. 802-5.
Cepko, C.L., S. Bruhn and D. Fekete, Methods in Cell Biology.
Rosenberg, W.S., X.O. Breakefield, C. DeAntonio and O. Isacson, Brain Res Mol Brain Res, 1992. 16(3-4): p. 311-5.
Zoellner, H.F. and N. Hunter, J Histochem Cytochem, 1989. 37(12): p. 1893-8.
Bao, Z.Z. and C.L. Cepko, J Neurosci, 1997. 17(4): p. 1425-34.
Snyder, E.Y., D.L. Deitcher, C. Walsh, S. Arnold-Aldea, E.A. Hartwieg and C.L. Cepko, Cell, 1992. 68(1): p. 33-51.
Moriyoshi, K., L.J. Richards, C. Akazawa, D.D. O'Leary and S. Nakanishi, Neuron, 1996. 16(2): p. 255-60. 29.
karnik, G., P.A. Negulescu, T.E. Knapp, L. Mere, N. Burres, L. Feng, M. Whitney, K. Roemer and R.Y. Tsien, Science, 1998. 279(5347): p. 84-8.
Cepko, C.L., C.P. Austin, C. Walsh, E.F. Ryder, A. Halliday and S. Fields-Berry, Cold Spring Harb Symp Quant Biol, 1990. 55: p. 265-78.
Turner, D.L., E.Y. Snyder and C.L. Cepko, Neuron, 1990. 4(6): p. 833-45.
Turner, D.L. and C.L. Cepko, Nature, 1987. 328(6126): p. 131-6.
Hughes, S.M. and H.M. Blau, Nature, 1990. 345(6273): p. 350-3.
Halliday, A.L. and C.L. Cepko, Neuron, 1992. 9(1): p. 15-26.
Muneoka, K., N. Wanek and S.V. Bryant, J Exp Zool, 1986. 239(2): p. 289-93.
Morgan, B.A. and D.M. Fekete, Methods Cell Biol, 1996. 51: p. 185-218.
Astrin, S.M., E.G. Buss and W.S. Haywards, Nature, 1979. 282(5736): p. 339-41.
Hamburger, V. and H.L. Hamilton, J Exp Morph, 1951. 88: p. 49-92.
Fekete, D.M. and C.L. Cepko, Mol Cell Biol, 1993. 13(4): p. 2604-13.
Spector, D.L., R.D. Goldman and L.A. Leinwand, Cells: a laboratory manual. 1997, Cold Spring Harbor, NY: Cold Spring Harbor Laboratories Press.
Walsh, C. and C.L. Cepko, Science, 1992. 255(5043): p. 434-40.
Walsh, C., C.L. Cepko, E.F. Ryder, G.M. Church and C. Tabin, Science, 1992. 258: p. 317-320.
Walsh, C. and C.L. Cepko, Nature, 1993. 362(6421): p. 632-5.
Reid, C.B., I. Liang and C. Walsh, Neuron, 1995. 15(2): p. 299-310.
Arnold-Aldea, S.A. and C.L. Cepko, Dev Biol, 1996. 173(1): p. 148-61.
Golden, J.A., S.C. Fields-Berry and C.L. Cepko, Proc Natl Acad Sci U S A, 1995. 92(12): p. 5704-8.
Golden, J.A. and C.L. Cepko, Development, 1996. 122(1): p. 65-78.
Szele, F.G. and C.L. Cepko, Curr Biol, 1996. 6(12): p. 1685-90.
Fields-Berry, S.C., in Genetics. 1990, Harvard University: Cambridge, MA. p. 141.
Ryder, E.F. and C.L. Cepko, Neuron, 1994. 12(5): p. 1011-28.
Sambrook, J., E.F. Fritsch and T. Maniatis, Molecular cloning: a laboratory manual. 1989, Cold Spring Harbor, NY: Cold Spring Harbor Laboratories Press.

上一页 [1] [2] [3] [4] [5] [6] [7] [8] [9] [10]

实验首页

实验论坛

实验软件

实验电书

实验视频

实验下载

仪器教程

文献检索

数据导航

生物公司

光盘刻录

实验助手

实验频道更新

实验论坛新帖

实验关注排行

相关实验文章

生物软件下载

仪器使用教程