Tet-on基因表達系統反應質粒PTRE-HIF-1α的構建和表達鑒定_《中國普外基礎與臨床雜志》

作者：

徐宗全 , 陳孝平 , 張萬廣 , 王其 , 關劍 , 李高鵬

華中科技大學同濟醫學院附屬同濟醫院肝臟外科中心(武漢430030);

關鍵詞：

低氧誘導因子-1α 巢式PCR Tet-on基因表達系統反應質粒克隆

視頻：

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目的克隆和構建攜帶人低氧誘導因子-1α（HIF-1α）的Tet-on基因表達系統反應質粒PTRE-HIF-1α。方法以缺氧的肝癌細胞株HepG2總RNA為模板，進行RT-巢式PCR，獲得HIF-1α的cDNA，克隆入Tet-on基因表達系統反應質粒PTRE2hyg，酶切重組子鑒定。將構建好的PTRE-HIF-1α用脂質體法轉入HepG2Tet-on細胞，在強力霉素的作用下，用RT-PCR及Western blot法鑒定重組質粒的表達。結果擴增出HIF-1α的cDNA測序結果與Genbank記載完全一致，并成功克隆入PTRE2hyg，將反應質粒PTRE-HIF-1α轉入HepG2Tet-on細胞，可以完整有效表達HIF-1α且受強力霉素的調控。結論成功克隆和構建攜帶HIF-1α基因的Tet-on基因表達系統反應質粒PTRE-HIF-1α，并證明其表達能在HepG2Tet-on細胞中受強力霉素調控。

引用本文： 徐宗全,陳孝平,張萬廣,王其,關劍,李高鵬. Tet-on基因表達系統反應質粒PTRE-HIF-1α的構建和表達鑒定. 中國普外基礎與臨床雜志, 2008, 15(12): 887-891. doi: 復制

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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. 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. 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. 受強力霉素調控表達的人肝癌HepG2細胞系的建立 [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.

《中國普外基礎與臨床雜志》

Tet-on基因表達系統反應質粒PTRE-HIF-1α的構建和表達鑒定

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《中國普外基礎與臨床雜志》

Tet-on基因表達系統反應質粒PTRE-HIF-1α的構建和表達鑒定

摘要 全文 圖表 視頻 參考文獻 施引文獻 補充材料

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Content

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