摘要
由白细胞介素2(IL-2)及其受体(IL-2R)为核心的自分泌体系在T淋巴细胞的正常生长、增殖和免疫功能的发挥中起着关键作用。高亲和力的白细胞介素2受体至少由α、β、γ三种亚基组成,其中只有α亚基呈诱导表达,而且是淋巴细胞中结合IL-2的唯一特异成分,因此,IL-2α亚基在机体对IL-2作用的免疫效应中起着重要作用,与其相一致,IL-2Rα亚基的表达受到严格的调控。
基因定向整合技术是新近发展起来可用于细胞基因组定向修饰的新方法。为了研究IL-2Rα基因在T淋巴细胞的功能,并建立体细胞基因敲除的模式系统,我们对在Jurkat细胞中敲除IL-2Rα基因进行了探索,并对IL-2Rα基因第一内含子的转录调控活性进行了初步研究。
一、悬浮细胞甲基纤维素半固体克隆化培养方法的建立与Jurkat Tet-on细胞系的建立。我们借鉴在杂交瘤细胞中用于克隆化培养的甲基纤维素半固体培养方法,用于悬浮细胞Jurkat细胞的克隆化培养,成功地建立了Jurkat Tet-on细胞系-rtTA③。通过Southern杂交显示,在rtTA③细胞中有rtTA基因的整合,Luciferase报告基因的转染结果表明,rtTA③细胞系在Dox 1μg/ml浓度下诱导近30倍的Luciferase的表达。
二,IL-2Rα基因第三内含子的克隆与核苷酸序列测定。应用PCR方法,扩增了IL-2Rα基因第三内含子2.5Kb片段,并成功克隆到pBS-SK载体中。构建了一系列亚克隆并测定了2.5Kb全核苷酸序列,其中2228bp碱基序列为国内外首次测定,此核苷酸序列已被Genbank接受,接受号为U56389。通过计算机分析发现IL-2Rα基因第三内含子具有典型的G↓GU……Py rich(~15bp)NCAG↓GU结构特点,
Interleukin 2 (IL-2), a key regulator of the immune response, is secreted by activated T cells upon antigenic stimulation. Binding of IL-2 to its high-affinity receptor (IL-2R ) induces proliferation as well as functional differentiation of T and B lymphocytes and natural killer activation. The high affinity IL-2 receptor is comprised of three component chains, IL-2Rα, IL-2Rβ, and IL-2Rγ. IL-2Rβ and IL-2Rγ are present constitutively in resting lymphocytes. In contrast, IL-2Rα is expressed only following activation and it is the unique subunit which strictly bind with IL-2. Therefore, the interleukin 2 receptor α-chain plays a critical role in IL-2 responsiveness, thus explaining why expression of this chain is highly regulated.
Homologous recombination is a powerful tool which has only recently become available for correcting or mutating the desired chromosomal locus. To establish the model of gene targeting in somatic cells and study the function of IL-2Rα gene in lymphocytes, we perform gene targeting of IL-2Rα in Jurkat cells and investigate the transcription regulation activity of the 1st intron of IL-2Rα gene.
I. Establishment of cell cloning in semi-solid medium in suspension cell and the construction of Jurkat Tet-on Cell line.
We successfully constructed Jurkat Tet-on cell line-rtTA(3) according to the protocol of hybridoma cloning culture using semi-solid medium of methyl cellulose. rtTA gene integrated into the chromosome of rtTA(3) cell line . These positive rtTA(3) cell line identified by Southern blot could express 30 folds luciferase highly while induced by 1 μg/ml of Dox.
引文
沈珝琲,方福德主编,1996 真核基因表达调控.高等教育出版社
莽锐,1995,中国协和医科大学硕士研究生毕业论文
邢光新,1996,中国协和医科大学博士研究生毕业论文
鄂征,1995,组织培养和分子细胞学技术.鄂征主编,北京出版社出版 p100-107.
孙乃恩,孙东旭,朱德煦编著 1991.分子遗传学。南京大学出版社 p404-469.
Bonton, W.D. et al, 1997. Science 196:180-182
Bueler,., et al 1993, Cell 73: 1339-1347
Cantrell, D.A., et al, 1983, J.Exp.Med, 158:1895
Capecchi, M.R.,1989, Science 244:1288-1292
Charron, J., et al, 1990. Mol. Cell. Biol. 10:1799-1804.
Chen,J., et al, 1994. Int. Immunol. 6:1265-1268
Cheng, Z. et al. 1994, PNAS, U.S.A. 91:5695-5699
Colombol, M. et al, 1994 Immunol Today, 15:48.
Davis, J.M. et al, 1982. J.Immunal.Methods 50:161
Deng, C. et al, 1992, Mol.Cell. Biol. 12:3365-3370
Depper, J.M. et al 1984. J. Immunol. 133: 3054
Domingues, O., et al, 1994 Nucl. Acids. Res. 22:3247-3248
Erlich, H.A., et al. 1991, Science 252:1643.
Feldhous, A.L., et al, 1993. The EMBO Journal 12 (7): 2763-2772.
Giri, J.G., et al, 1995, EMBO J. 14:3654
Gossen, M. et al 1992. Proc.Natl.Acad.Sci.USA89:5547-5551
Gossen, M., et al. 1995. Science 268: 1966-1769.
Green, W.C. et al. 1985, J.Exp. Med. 162:363
Greene, W.C, et al. 1989. Immunol.Today 10:272Guartowente, L. et al, 1995. Trends. Biochem. Sci. 20: 517
Harlow, E. et al, 1988. Antibodies: A Laboratory Manual, page219, Cold Spring Harbor Laboratory
Hasty, P. et al, 1991. Mol. Cell. Biol. 11:5586-5591.
Hatakeyama, M.M., et al. 1989 Science 244:551
Hattori,N. et al, 1996. J.Exp. Med. 184:1137-1147
Heikoffer, S. 1987. Methods in Enzymology 155;156-165
Horiuchi, S, et al. 1997. Immunology. 91(1): 28-34
IBI corporation. An application protocol for underetioned eletroelter IBI model UEA.
John,S., et al, 1995. Mol.Cell.Biol. 15: 1786-1796
Joyer, A.L., et al, 1989. Nature 338:153-156
Kaslow, D.C. 1986 Nucleic Acid. Res. 14:67
Koller, B.H. et al, 1992, Annu Rev Lmmunop: 10:705
Kou H-C, et al, 1991, Science 251-1045
Krainer, A.R., et al, 1990 Cell 62:35
Kronke,M. et al. 1985. Science 228:1215
Kuang, A.A., et al, 1993, Mol.Cell.Biol. 12:2536-2545
L. Feldhaus, A., et al, 1993 the EMBO J. 12:2763-2772.
Lecine, P., et al. 1996, Mol.Cell.Biol. 16: 6829-6840.
Leonard, W.J. 1991. Nature 253: 858-61
Leonard, W.J. 1992 InJ. Wawman and F.Balkwill (ed.) Interleukin-2 Blockwell Scientific Publication Ltd, Oxford.
Leonard, W.J. et al. 1984 Nature 311:626
Leonard, W.J., et al, 1994. Immunol.Rev. 138:61
Leonard, W.J., et al. 1985a. Science, 230:633
Leonard, W.J, et all 1985b. PNAS. U.S.A. 82: 6281
Lin, B.B. et al., 1990, Mol.Cell.Biol: 10: 850-853
Maniatis, T, et al., 1987. Science 236: 1237
Mansour, S.K. et al, 1988. Nature 336:348-352.
Morgan, D.A. et al, 1976. Science 193: 1007
Mortensen, R.M., et al, 1992. MCB12 (5): 2391-2395
Nikaido, T., et al, 1984 Nature 311:631.
Ochman, H., et al, 1988.Genetics 120:621-623
Pierce, J.W., et al, 1995, J. Immunol., 155: 1972-1980
Ponticelli, A.S. et al, 1985. Cell 41:145-151
Preparation of plasmid DNA: A modified Mini Alkaline-lysis/PEG. Provided with the Taq didexy Terminator Cycle Sequencing kit. (ABI/N 4011507)
Promega 公司, Prime-a-Gene~N Labeling System 说明书
Prusiner, .B. et al. 1990 Cell 63: 673-686
Rolink,A., et al., 1994. Int. Immunol., 6:1257-1264.
Romeo, P.H., 1997. C.R. Seaces SocBiol.Fil. 191:105-111.
Rosenthal, A., 1992 Cloning techniques, 10:44-48.
Rothenberg, E.V., 1992. Adv. Immunol. 51: 86-214
Sambrook, J., et al. 1989. Molecular Cloning, A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press
Schorle, H., et al., 1991. Nature 352:621-624
Sharp, P.A. 1994, Cell, 77: 805-815
Shen, Y., et al. 1997, FEBS. 413: 92-98
Shesely, E.G., et al, 1991, PNAS. U.S.A. 88:4294-4298.
Smith, C.W.J. et al, 1989 Cell 56:749
Smith, K.A. 1984, Annu.Rev. Immunol 92:121
Smith, K.A. 1987 Immunol. Today 8: 11
Smith, K.A. 1988 science, 240: 1169-76
Smithies, O., et al. 1985, Nature 317:230-234
Swain, S.L. et al., 1990 J.Immunol. 144:4712
Takeshita, T. et al. 1992 Science 257: 379
Tanignchi, T. et al, 1993, Cell. 73:5-8
Taniguchi, T. 1995. Sceince 268: 251-255
teRride, H., et al., 1992, PNAS. U.S.A. 89: 5128-5132
Theze, J. et al, 1996 Immunol.Today 17 (10):481
Thomas, K.R. and Capecchi, M.R. 1986. Nature 324:34-38
Thomas, K.R. et al, 1986 Cell. 44:419-428.
Thomas, K.R., et al, 1987. Cell. 51:503-512.
Thomas, K.R., et al, 1992. Mol.Cell.Biol. 12: 2919-2923
Thuy,L.T.B. et al, 1987. J.Immunol. 139: 1150
Ting, CN., et al, 1996. Nature 384:474-478
Tjian, R.et al., 1994 Cell 77:5
Tsudo, M.T., et al, 1984 J.Exp.Med 160:612
USB 公司, Sequenase Version 2.0 说明书
Waldman,T.A., 1986 Science 240 :1169.
Waldmann, T.A., et al, 1989, Annu.Rev. Biochem. 58:875
Willerford, D.M., et al, 1995. Immunity 3: 521-530
Yamagata, T. et al, 1997. Leukemia, 11 suppl 3:501-502.
Yu, J.S., et al. 1996. Cancer Res. 56: 5423-5427
Zawel, L.et al, 1995. Annu.Rev.Biochem. 64:533
Zhen, L. and Swank, R.T. 1993. Biotechniques 4(6): 151
Abremski, K. and Hoess, R.H. 1984. Baetenophage P1 site-specific recombination: Purification and properties of the Cre recombinase protein. J. Biol. Chem. 259:1509-1514;
Adra, C.N. et al. 987. Cloning and expression of the mouse pgk-1 gene and the nucleotide sequence of its promoter Gene, 60:67-74;
Askew, G.R. et al. 1993. Site-directed point mutations in embryonic stem cells: a gene-targeting tag and exchange strategy. Mol. Cell. Biol. 13:4115-4124;
Austin, S. et al. 1981. A novel role foe site-specific recombination in maintenance of bacterial replicons. Cell 25:729-736;
Bradley, A., et al. 1984. Formation of germ-line chimaeras from embryo-derived teratocarcinoma cell lines. Nature 309:255-256;
Brandon, E.P. et al. 1995. Targeting thew mouse genome: a compendium of knockout (part Ⅰ). Curr Biol. 5:625-634;
Brandon, E.P. et al. 1995. Targeting thew mouse genome: a compendium of knockout (part Ⅱ). Curr. Biol. 5:758-768;
Brandon, E.P. et al. 1995. Targeting thew mouse genome: a compendium of knockout (part Ⅲ). Curr. Biol. 5:873-1073;
Capecchi MR. 1989. Altering the genome by homologous recombination. Science 244:1288-1292;Capecchi, M.R. 1980, High efficiency transformation by direct microinjection of DNA into cultured mammalian cells. Cell, 22:479;
Charron, J. et al. 1990. High-Frequency disruption of the N-myc Gene in embryonic stem and Pre-B lines by homologous recombination. Mol. Cell. Biol. 10:1799-1804;
Copp, A.J. 1995. Death before birth: clues from gene knockout and mutations. Trends Gnet, 11:87-93;
Danos, O. et al. 1988 Safe and efficient generation of recombinant retroviruses with amphotropic and ecotropic host ranges. Proc. Natl. Acad. Sci. U.S.A. 85:6460-6464;
Deng C. Capecchi M. 1992. Reexamination of gene targeting frequency as a function of the extent of homology between the targeting receptor and the target locus. Mol. Cell. Biol. 12:3365-3371;
Disanto,J.P. et al. 1995. Lymphoid development in mice with a targeted deletion of the interleukin-2 receptor a china. Proc. Natl. Acad. Sci. U.S.A. 92:377-381;
Doetschman, T. et al. 1987. Nature 330:576;
Donehower, P.A. Mice deficient for 53P are developmentally normal but susceptible to spontaneous tumors. Nature, 356:215-221;
Evans, M.J., and kaufman, M.H. 1981. Establishment in culture of pluripoptential cells from mouse embryos. Nature. 292:154-156;
Feil, P. et al. 1996. Ligand-activated site-specific recombination in mice. Proc. Natl. Acad. Sci. U.S.A. 93:10887-10890;
Feldhaus, A.L. et al 1993. Targeted disruption of the Oct-2 locus in a B cell provides genetic evidence for two distinct cell type-specific pathways of octamer element-mediated gene activation.
Goode now, R.S. et al 1983. Nature 301:388-394;
Gossen, M. et al. 1995. Transcriptional activation by tetracyclines in mammalian cells. Science. 268:1766-1769;
Gridely,T. 1991. Insertional versus trgeted mutagenesis in mice. The New Biologist 3: 1025-1034;
Gritz. L. et al. 1983. Plasmid encoded hygrymycin B resistance: the sequence of hygromicin B phosphotransferase gene and its expression in Eschenchia coli and Saccharomyces cerevisiae. Gene 25:179-188;
Gu, H. et al. 1994. Deletion of a DNA polymerase b-gene Segment in T cell using cell-typespecific gene targeting. Science 265:103-106;
Gu, H.I. et al. 1993. Independent control of immunoglobulin swith recombination at individual switch regions evidenced through Cre-loxP-mediated gene targeting. Cell 73:1155-164;
Hamilton, D.L. and Abremski, K. 1984. Site-specific recombination by the bacterialophage PI lox-Cre system: Cre-mediated Synapsis of two lox site. J. Mol. Biol. 178:481-486;
Hanks, M. et al. 1995. Rescue of the En-1 mutant phenotype by replacement of En-1 with En-2. Science 269:679-682;
Hasty, P. et al 1991 Target frequency and intergration pattern for insertion and replacement vectors in embryonic stem cells, mol. Cell. Biol. 11:4509-17;
Hasty, P. et al. 1991. Introduction of subtle mutation into the Hox-2.6 locus in embryonic stem cells. Nature 350: 243-246;
Hasty, P. et al. 1991. THe length of homology required for genbe targeting in embryonic stem cells. Mol. Cell Biol 11:5586-5591;
Hasty, P. et al. 1992. THe role and fate of DNA ends for homologous recombination in embryonic stem cells. Mol. Cell Biol. 12: 2464-2474;
Hinen A. et al. 1978. PNAS 75:1929;
Hoes, R.H. et al. 1986 The role of the lox P spacer region in P1 site-specific recombination. Nucleic Acids Res. 14:2287-2230;
Hoess R.H. and Abremski, 1984. Interaction of the bacteriophage P1 recombinase Cre with the recombimng site loxp. Proc. Natl. Acad. Sci. U.S.A. 81:1026-1029;
Hoess, R.H. et al. 1982. P1 site-specific recombination: nucleotide sequence of the recombimng site. Proc. Natl. Acad. Sci. U.S.A. 79:3398-3402;Hoess. R.H. et al. 1984. The nature of the interaction of the P1 recombinase Cre with the recombining site loxP. Cold Spring Harbor Symp. Quant. Biol. 49:761-768.
Hooper. M. et al 1987. Nature 326:292;
Ishiura M. et al 1982, Phage partial-mediated gene transfer to cultured mammalian cell. Mol. Cel. Biol. @:607-616;
Jiang. RL. and Gndley, T. 1997. Gene targeting: Things go better with CRE. 7: R321-323;
Joyner, et al. 1989. Production of a mutation in En-2 gene by homologous recombination in embryonic stem cells. Nature 338:153-156;
Kuhn,R. et al. 1995. Inducible gene targeting in mice Science 269:1427-1429;
Kim. Hyang-Swk, et al. 1988. Rcombinant fragment assay for gene targetting based on the polymmerase chain reaction. Nucl. Acid. Res. 16(18):8887-8903;
Kistner, A. et al. 1996. Doxycline-mediated quantative and tissue-specific control of gene expression in transgenic mice. Proc. Natl. Acad. Sci. U.S.A. 93: 10933-10938;
Koller, B.H. & Smithies,O. 1992, Annu Rev Immunop. 10: 705;
Lakso, M. et al. 1992. Targeted oncogene activition by site-specific recombination in transgenic mice. Proc. Natl. Acad. Sci. U.S.A. 89:6232-236;
Lesch, et al. 1964. A familial disorder of Uric acid metabolism and central nervous system function. Am. J. Med. 36:561-570;
Lewis, J. et al 1996. Gene modification via "Plug and Socket" gene targeting. J. Clin. Invest. 97(1):3-5;
Li, E. 1993, Normal development and growth of mice carrying a targeted disruption of the a -lretinoic acid receptor gene. Proc. Natl. Acad. Sci. U.S.A. 90: 1590-1594;
Llllankenau, DN. 1995. Genetics of genetics in Drosophila: P elements serving the study of homologous recombination, gene conversion and targeting. Ohromosoma 103:659-668;Mansour, S.KL. et al. 1988, Disruption of the proto-oncogen int-2 in mouse embryo-derived stem cells: a strategy for targeting mutation to nonselectable genes. Nature 336:348-352;
Mattinm, G. 1981. Isolation of a pluripoptential cell line from early mouse embryos culture i medium conditioned by teratocarcinoma stem cells.Proc. Natl. Acad. Sci. U.S.A. 78:7634-7638;
Melton, D.W. 1994. Gene targeting in the mouse. BioEssays 16:633-638;
Moeus, C.B. 1993. Defects in heart and lung development in compound heterozygotes for two different tyargeted mutationsa at the N-myc locus. Development. 119:485-499;
Morgenstern, J.P. et al. 1990. Advanced mammalian gene transfer: High-titer retroviral vectors with multiple drug selection markers and a complementary helper-free lackaging cell line. Nucl. Acids. Res. 18:3587-3596;
Mulligan, R.C. 1993. The Basic science of gene therapy Science 260:926-932
Neumann, E. et al. 1982, Gene transfer into mouse lyoma cells by electroporation in high electric fields. EMBO J.I:841;
Orban, PC. et al. 1992. Tissue and site- specific DNA recombination in transgenic mice. Proc. Natl. Acad. Sci. U.S.A. 89: 6861-6865;
Orr-weaver, T.L. et al. 1981. PNAS 78:6354;
Reid, L.H. et al. 1991. Cotransformation and gene targeting in mouse embryonic stem cells. Mol. Cell. Biol. 11:2769-2777;
Rossant, J. 1991. Gene disruption in mam#ds. Curr.Opinion Genet. Devel. 1:236-240;
Rothstein, R.J. et al. 1983. Methods Enzymol 101:202;
Saiki, R.K. et al. 1985. Enzymatic amplification of # globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230: 1350-1354;
Sauer, B. and Henderson, N. 1988. Site-specific DNA recombination in mammalian cells by the Cre recombmase of bacterialophage P1. Proc. Natl. Acad. Sci. U.S.A. 85:5166-5170;Sauer, B. and Henderson, N. 1989. Cre-stimulated recombination at lox P containing DNA sequences placed into the mammalian genome. Nucleic Acids. Res. 17:147-161;
Sauer, B. and Henderson, N. 1990. Targeted insertion of exogenous DNA into the enkaryotic genome by the Cre recombinase. New Biol. 2:441-449.
Schwartz berg, PL. et al. 1990. Targeted gene disruption the endogenous c-abl Locus by homologous recombination with DNA encoding a selectable fusion protein. Proc. Natl. Acad. Sci. U.S.A. 87:3210-3214;
Schwartz berg, PL. et al, 1989. Germline transmission of a c-abl mutation produced by targeted gene disruption in ES cells. Science 246:799-803;
Sedivy, J.M. and Sharp, P.A. 1989. Positive genetic selection foe gene disruption in mammalian cells by homologous recombination. Proc. Natl. Acad. Sci. U.S.A. 86:227-231;
Serian, P. 1995 Gene targeting in ES cells. Annu. Rev. Nerpsci., 18:1-18;
Shesely, E.G. et al. 1991. Correction of a human b~s-globin gene by gene targeting. Proc. Natl. Acad. Sci. U.S.A. 88:4294-4298;
Smith, G.R. 1987. Mechanism and control of homologous recombination in Escherichia coli. Ann. Rev. Genetics 21:179-201;
Smithies, O. et al. 1985. Insertion of DNA sequences into the human chromosome #globin by homologous recombination. Nature 317, 230-234;
Soriano, P. et al. 19912. Targeted disruption of the C-src prote-oncogene leads to osteopetrosis in mice. Cell 64: 693-702;
Stahl, F.W. 1987. Scientific American 256
Te Riele, H. et al. 1990. Consecutive inactivation of both allels of the pim-1 proto-oncogene by homologous recombination in embryonic stem cells. Nature, 348:649-651;
Thomas, K. R. and Capecchi, M.R. 1986. Introduction of homologouse DNA sequences into mammalian cells induces mutations in the cognate gene. Nature 324:34-38;
Thomas, K. R et al. 1996. High frequency targeting of genes to specific sites in the mammalian genome Cell. 44: 419-428
Thomas, K.R. et al. 1986. high frequency targeting of genes to specific sites in the mammalian genome. Cell 44:419-428;
Thomas, K.R. el al. 1987. Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell. 51:503-512;
Thompson, CJ. et al. 1983 Nucleotid sequence of a streptomycete aminoglycoside phosphotransferase gene and its relationship to phosphotransforases encoded by resistance plasmids. Proc. Natl. Acad. Sci. U.S.A. 80:5190-5194;
Threadgill,D.W. 1995. Targeted disruption of mouse EGF receptor: effect of genetic background on mutant phenotype. Science, 269:230-234;
Torres, R.M. et al. 1996 Aberrant B cell development and immune response in mice with a compromised BCR complex. Science (Wash. DC). 272:1804-1808;
Tsien, J.I. et al. 1996. Subregion and cell type-restricted gene knockout in mouse brain. Cell 87:1317-1326;
Valancius, V. and Smithies, O. 1991. Testing an "in-out" targeting procedure for making subtle genomic modifications in mouse embryonic stem cells. Mol. Cell. Biol. 11.1402-1408;
Williams, R. et al. Gene targeting in normal diploid human cells. Yoshie,O. et al.1984 PNAS. 81:649-653;
Zimmer man V. 1982, Eletric field-mediated fusion and related electrical phenomena. Biochim. Biophys. Acta 694:227;
Zimmer, A. et al 1989 Production of chimaeric mice containing embryonic stem (ES) cells carrying a homoebox Hoxl.1 allel mutated by homologous recombination Nature. 338:150-153;
Zimmer, A. el al. 1991. Production of chimaeric mice containing embryonic stem (ES) cells carrying a homobox Hoxl.l allel mutated by homologous recombination. 338:150-153.