Construction and Expression Analysis of Recombinant Vector PTRE-HIF-1α of Tet-on Gene Expression System_CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Authors：

XU Zongquan , CHEN Xiaoping , ZHANG Wanguang , WANG Qi , GUAN Jian , LI Gaopeng

Hepatic Surgery Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China;

Corresponding author：

Keywords：

Hypoxia-inducible factor-1α; Nested PCR; Tet-on gene expression system; Responsive plasmid; Cloning

Video：

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Objective To construct the responsive plasmid PTRE-HIF-1αof Tet-on gene expression system and examine its expression. Methods RT-nested PCR was performed on the total RNA extracted from hypoxia HepG2 cells to obtain the cDNA of HIF-1α, which was inserted into the responsive plasmid PTRE2hyg. DNA sequencing was performed after the recombinant of responsive plasmid PTRE-HIF-1α was identified by endonuclease digestion. This recombinant vector was transfected into HepG2Tet-on cells by means of liposome and its expression was examined by RT-PCR and Western blot under the control of deoxycycline. Results The amplified products were confirmed as the cDNA of HIF-1α by DNA sequencing. The responsive plasmid PTRE-HIF-1α verified by edonuclease digestion, was capable of expression in HepG2Tet-on cells and could be controlled by deoxycycline. Conclusion The responsive plasmid PTRE-HIF-1α of Tet-on expression system is constructed successfully, and it can express under the regulation of deoxycycline in the HepG2Tet-on cells.

Citation： XU Zongquan,CHEN Xiaoping,ZHANG Wanguang,WANG Qi,GUAN Jian,LI Gaopeng,.. Construction and Expression Analysis of Recombinant Vector PTRE-HIF-1α of Tet-on Gene Expression System. CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY, 2008, 15(12): 887-891. doi: Copy

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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. 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. 李东华, 陈孝平, 全晓明. 受强力霉素调控表达的人肝癌HepG2细胞系的建立 [J]. 中华普通外科杂志, 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.

CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Construction and Expression Analysis of Recombinant Vector PTRE-HIF-1α of Tet-on Gene Expression System

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