• 華中科技大學(xué)同濟醫(yī)學(xué)院附屬同濟醫(yī)院肝臟外科中心(武漢430030);

目的  克隆和構(gòu)建攜帶人低氧誘導(dǎo)因子-1α（HIF-1α）的Tet-on基因表達系統(tǒng)反應(yīng)質(zhì)粒PTRE-HIF-1α。方法  以缺氧的肝癌細(xì)胞株HepG2總RNA為模板，進行RT-巢式PCR，獲得HIF-1α的cDNA，克隆入Tet-on基因表達系統(tǒng)反應(yīng)質(zhì)粒PTRE2hyg，酶切重組子鑒定。將構(gòu)建好的PTRE-HIF-1α用脂質(zhì)體法轉(zhuǎn)入HepG2Tet-on細(xì)胞，在強力霉素的作用下，用RT-PCR及Western blot法鑒定重組質(zhì)粒的表達。結(jié)果  擴增出HIF-1α的cDNA測序結(jié)果與Genbank記載完全一致，并成功克隆入PTRE2hyg，將反應(yīng)質(zhì)粒PTRE-HIF-1α轉(zhuǎn)入HepG2Tet-on細(xì)胞，可以完整有效表達HIF-1α且受強力霉素的調(diào)控。結(jié)論  成功克隆和構(gòu)建攜帶HIF-1α基因的Tet-on基因表達系統(tǒng)反應(yīng)質(zhì)粒PTRE-HIF-1α，并證明其表達能在HepG2Tet-on細(xì)胞中受強力霉素調(diào)控。

引用本文： 徐宗全,陳孝平,張萬廣,王其,關(guān)劍,李高鵬. Tet-on基因表達系統(tǒng)反應(yīng)質(zhì)粒PTRE-HIF-1α的構(gòu)建和表達鑒定. 中國普外基礎(chǔ)與臨床雜志, 2008, 15(12): 887-891. doi: 復(fù)制

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2. Semenza GL. HIF-1 and human disease: one highly involved factor [J]. Genes Dev, 2000; 14(16)∶1983.
3. 李東華, 陳孝平, 全曉明. 受強力霉素調(diào)控表達的人肝癌HepG2細(xì)胞系的建立 [J]. 中華普通外科雜志, 2004; 19(4)∶225.
4. Semenza GL, Rue EA, Iyer NV, et al. Assignment of the hypoxia-inducible factor 1 alpha gene to a region of conserved synteny on mouse chromosome 12 and human chromosome 14q [J]. Genomics, 1996; 34(3)∶437.
5. Sambrook J, Fritsh EF, Maniatios T, et al Eds. Molecular cloning. A laboratory manual [M]. New York: Cold Spring Harbor Laboratory, 1989∶16-69.
6. Gossen M, Bujard H. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters[J]. Proc Natl Acad Sci USA, 1992; 89(12)∶5547.
7. Gossen M, Freundlieb S, Bender G, et al. Transcriptional activation by tetracyclines in mammalian cells[J]. Science, 1995; 268(5218)∶1766.
8. Shen Q, Sun Q, Wei X, et al. Generation and characterization of islet cell tumor in pTet-on/pTRE-SV40Tag double-transgenic mice model [J]. J Biosci Bioeng, 2007; 104(1)∶14.
9. Das AT, Klaver B, Harwig A, et al. Construction of a doxycycline-dependent simian immunodeficiency virus reveals a nontranscriptional function of tat in viral replication[J]. J Virol, 2007; 81(20)∶11159.
10. Hojman P, Eriksen J, Gehl J. Tet-On induction with doxycycline after gene transfer in mice: sweetening of drinking water is not a good idea[J]. Anim Biotechnol, 2007; 18(3)∶183.
11. Huai J, Firat E, Niedermann G. Inducible gene expression with the Tet-on system in CD4+ T cells and thymocytes of mice[J]. Genesis, 2007; 45(7)∶427.
12. Watanabe T, Saito D, Tanabe K, et al. Tet-on inducible system combined with in ovo electroporation dissects multiple roles of genes in somitogenesis of chicken embryos[J]. Dev Biol, 2007; 305(2)∶625.
13. Zhou X, Symons J, Hoppes R, et al. Improved single-chain transactivators of the Tet-On gene expression system[J]. BMC Biotechnol, 2007; 7∶6.
14. Zhou X, Vink M, Berkhout B, et al. Modification of the Tet-On regulatory system prevents the conditional-live HIV-1 variant from losing doxycycline-control [J]. Retrovirology, 2006; 3∶82.
15. Shaikh S, Nicholson LF. Optimization of the Tet-On system for inducible expression of RAGE[J].J Biomol Tech, 2006; 17(4)∶283.
16. Wang Y, Cong X, Liu D, et al. The transfection of embryonic stem cells with Tet-on system and its responsiveness to doxycycline [J]. Sci China C Life Sci, 2005; 48(4)∶307.
17. Giménez E, Lavado A, Giraldo P, et al. A transgenic mouse model with inducible Tyrosinase gene expression using the tetracycline (Tet-on) system allows regulated rescue of abnormal chiasmatic projections found in albinism [J]. Pigment Cell Res, 2004; 17(4)∶363.
  1. 1. Wang GL, Jiang BH, Rue EA, et al. Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension [J]. Proc Natl Acad Sci USA, 1995; 92(12)∶5510.
  2. 2. Semenza GL. HIF-1 and human disease: one highly involved factor [J]. Genes Dev, 2000; 14(16)∶1983.
  3. 3. 李東華, 陳孝平, 全曉明. 受強力霉素調(diào)控表達的人肝癌HepG2細(xì)胞系的建立 [J]. 中華普通外科雜志, 2004; 19(4)∶225.
  4. 4. Semenza GL, Rue EA, Iyer NV, et al. Assignment of the hypoxia-inducible factor 1 alpha gene to a region of conserved synteny on mouse chromosome 12 and human chromosome 14q [J]. Genomics, 1996; 34(3)∶437.
  5. 5. Sambrook J, Fritsh EF, Maniatios T, et al Eds. Molecular cloning. A laboratory manual [M]. New York: Cold Spring Harbor Laboratory, 1989∶16-69.
  6. 6. Gossen M, Bujard H. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters[J]. Proc Natl Acad Sci USA, 1992; 89(12)∶5547.
  7. 7. Gossen M, Freundlieb S, Bender G, et al. Transcriptional activation by tetracyclines in mammalian cells[J]. Science, 1995; 268(5218)∶1766.
  8. 8. Shen Q, Sun Q, Wei X, et al. Generation and characterization of islet cell tumor in pTet-on/pTRE-SV40Tag double-transgenic mice model [J]. J Biosci Bioeng, 2007; 104(1)∶14.
  9. 9. Das AT, Klaver B, Harwig A, et al. Construction of a doxycycline-dependent simian immunodeficiency virus reveals a nontranscriptional function of tat in viral replication[J]. J Virol, 2007; 81(20)∶11159.
  10. 10. Hojman P, Eriksen J, Gehl J. Tet-On induction with doxycycline after gene transfer in mice: sweetening of drinking water is not a good idea[J]. Anim Biotechnol, 2007; 18(3)∶183.
  11. 11. Huai J, Firat E, Niedermann G. Inducible gene expression with the Tet-on system in CD4+ T cells and thymocytes of mice[J]. Genesis, 2007; 45(7)∶427.
  12. 12. Watanabe T, Saito D, Tanabe K, et al. Tet-on inducible system combined with in ovo electroporation dissects multiple roles of genes in somitogenesis of chicken embryos[J]. Dev Biol, 2007; 305(2)∶625.
  13. 13. Zhou X, Symons J, Hoppes R, et al. Improved single-chain transactivators of the Tet-On gene expression system[J]. BMC Biotechnol, 2007; 7∶6.
  14. 14. Zhou X, Vink M, Berkhout B, et al. Modification of the Tet-On regulatory system prevents the conditional-live HIV-1 variant from losing doxycycline-control [J]. Retrovirology, 2006; 3∶82.
  15. 15. Shaikh S, Nicholson LF. Optimization of the Tet-On system for inducible expression of RAGE[J].J Biomol Tech, 2006; 17(4)∶283.
  16. 16. Wang Y, Cong X, Liu D, et al. The transfection of embryonic stem cells with Tet-on system and its responsiveness to doxycycline [J]. Sci China C Life Sci, 2005; 48(4)∶307.
  17. 17. Giménez E, Lavado A, Giraldo P, et al. A transgenic mouse model with inducible Tyrosinase gene expression using the tetracycline (Tet-on) system allows regulated rescue of abnormal chiasmatic projections found in albinism [J]. Pigment Cell Res, 2004; 17(4)∶363.