四环素诱导调控DEC1表达的Vero细胞系的构建及鉴定
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摘要
为了构建四环素(tetracycline,Tet)诱导的利用Tet-on诱导表达系统调控的Vero-TR母细胞,建立Tet诱导表达或沉默DEC1的Vero细胞系并加以鉴定。试验将表达Tet阻遏蛋白(Tet repressor protein,TetR)的pc DNA6转染至Vero细胞,通过Blasticidin筛选稳定表达TetR的阳性细胞克隆,即Vero-TetR母细胞系。将克隆有DEC1基因(CDS区的pc DNA4真核表达载体或克隆有特异性靶向DEC1基因的shRNA的p Babe-H1真核表达载体,分别转染至Vero-TR细胞,通过Zeocin或Puromycin筛选诱导表达或沉默DEC1的阳性细胞克隆,命名为Vero-DEC1和VerosiDEC1。利用Western blot检测Vero细胞中Tet诱导表达或沉默DEC1的效果。最后成功构建了Vero-TR、Vero-DEC1和Vero-siDEC1的稳定细胞系,Western blot结果显示DEC1能够在Tet的调控下有效表达或沉默。Vero-TR母细胞系的成功构建为建立其他宿主细胞基因表达或沉默的细胞系提供了重要材料,Vero-DEC1和Vero-siDEC1细胞系的成功构建为研究DEC1在正常或感染等应激情况下的生物学功能奠定了基础。
This study aimed to generate tetracycline-induced Vero-TR parent cell line using Tet-on inducible system,and then to generate Vero cell lines that inducibly express or knock down DEC1. pc DNA6 expressing Tet repressor protein( TetR) was transfected into Vero cells,and positive clones that stably express TetR were selected by Blasticidin and designated Vero-TR parental cell line. In addition,pc DNA4 expressing DEC1 or the pBabe-H1 expressing DEC1 shRNA were transfected into Vero-TR,respectively. The positive cell clones that inducibly express or knockdown DEC1 were screened by Zeocin or Puromycin,and designated Vero-DEC1 or Vero-siDEC1. Moreover,the efficacy of expression or knockdown of DEC1 was detected by Western blot analysis. The stable cell lines of Vero-TR,Vero-DEC1 and Vero-siDEC1 were successfully generated. In addition,the Western blot results showed that DEC1 can be induced by tetracycline to express or knock down. The Vero-TR parental cell line will provide an important material for generating cell lines that inducibly express or knock down any other host cell genes. The Vero-DEC1 and Vero-siDEC1 cell lines obtained in this study lay a critical foundation for studying biological functions of DEC1 under normal or stress conditions of hosts.
This study aimed to generate tetracycline-induced Vero-TR parent cell line using Tet-on inducible system,and then to generate Vero cell lines that inducibly express or knock down DEC1. pc DNA6 expressing Tet repressor protein( TetR) was transfected into Vero cells,and positive clones that stably express TetR were selected by Blasticidin and designated Vero-TR parental cell line. In addition,pc DNA4 expressing DEC1 or the pBabe-H1 expressing DEC1 shRNA were transfected into Vero-TR,respectively. The positive cell clones that inducibly express or knockdown DEC1 were screened by Zeocin or Puromycin,and designated Vero-DEC1 or Vero-siDEC1. Moreover,the efficacy of expression or knockdown of DEC1 was detected by Western blot analysis. The stable cell lines of Vero-TR,Vero-DEC1 and Vero-siDEC1 were successfully generated. In addition,the Western blot results showed that DEC1 can be induced by tetracycline to express or knock down. The Vero-TR parental cell line will provide an important material for generating cell lines that inducibly express or knock down any other host cell genes. The Vero-DEC1 and Vero-siDEC1 cell lines obtained in this study lay a critical foundation for studying biological functions of DEC1 under normal or stress conditions of hosts.
引文
[1]Li Y,Xie M,Song X,et al.DEC1 negatively regulates the expression of DEC2 through binding to the E-box in the proximal promoter[J].J Biol Chem,2003,278(19):16899-16907.
[2]St-Pierre B,Flock G,Zacksenhaus E,et al.Stra13 homodimers repress transcription through class B E-box elements[J].J Biol Chem,2002,277(48):46544-46551.
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[7]Qian Y J,Zhang J,Jung Y S,et al.DEC1 coordinates with HDAC8to differentially regulate TAp73 and Delta Np73 expression[J].PLo S One,2014,9(1):e84015.
[8]Yamada K,Miyamoto K.Basic helix-loop-helix transcription factors,BHLHB2 and BHLHB3;their gene expressions are regulated by multiple extracellular stimuli[J].Front Biosci,2005,10:3151-3171.
[9]Fuyuki S,Ujjal K Bhawal,Tomohiro Yoshimura,et al.DEC1 and DEC2 crosstalk between circadian rhythm and tumor progression[J].J Cancer,2016,7(2):153-159.
[10]Sato F,Bhawal U K,Yoshimura T,et al.DEC1/STRA13/SHARP2and DEC2/SHARP1 coordinate physiological processes,including circadian rhythms in response to environmental stimuli[J].Curr Top Dev Biol,2014,110:339-372.
[11]Honma S,Kawamoto T,Takagi Y,et al.Dec1 and Dec2 are regulators of the mammalian molecular clock[J].Nature,2002,419(6909):841-844.
[12]Qian Y J,Zhang J,Yan B,et al.DEC1,a basic helix-loop-helix transcription factor and a novel target gene of the p53 family,mediates p53-dependent premature senescence[J].J Biol Chem,2008,283(5):2896-2905.
[13]Sun H,Lu B,Li R Q,et al.Defective T cell activation and autoimmune disorder in Stra13-deficient mice[J].Nat Immunol,2001,2(11):1040-1047.
[14]Miyazaki K,Miyazaki M,Guo Y,et al.The role of the basic helixloop-helix transcription factor Dec1 in the regulatory T cells[J].J Immunol,2010,185(12):7330-7339.
[15]Lin C C,Bradstreet T R,Schwarzkopf E A,et al.Bhlhe40 controls cytokine production by T cells and is essential for pathogenicity in autoimmune neuroinflammation[J].Nat Commun,2014,5:3551.
[16]Kanda M,Yamanaka H,Kojo S,et al.Transcriptional regulator Bhlhe40 works as a cofactor of T-bet in the regulation of IFNgamma production in i NKT cells[J].Proc Natl Acad Sci U S A,2016,113(24):E3394-3402.
[17]Bhawal U K.IL-1beta-mediated up-regulation of DEC1 in human gingiva cells via the Akt pathway[J].J Cell Biochem,2012,113(10):3246-3253.
[18]Ivanova A V,Ivanov S V,Zhang X,et al.STRA13 interacts with STAT3 and modulates transcription of STAT3-dependent targets[J].J Mol Biol,2004,340(4):641-653.
[19]Annamalai T,Saif L J,Lu Z,et al.Age-dependent variation in innate immune responses to porcine epidemic diarrhea virus infection in suckling versus weaned pigs[J].Vet Immunol Immunopathol,2015,168(3-4):193-202.
[20]Cecere T E,Todd S M and Leroith T.Regulatory T cells in arterivirus and coronavirus infections:do they protect against disease or enhance it[J].Viruses,2012,4(5):833-846.
[21]Charley B,Riffault S and Van Reeth K.Porcine innate and adaptative immune responses to influenza and coronavirus infections[J].Ann N Y Acad Sci,2006,1081:130-136.
[22]Nikolay A,Castilho L R,Reichl U,et al.Propagation of Brazilian zika virus strains in static and suspension cultures using vero and BHK cells[J].Vaccine,2017,S0264-410X(17):30324-30329.
[23]Castrillon-Betancur J C and Urcuqui-Inchima S.Overexpression of miR-484 and miR-744 in Vero cells alters dengue virus replication[J].Mem Inst Oswaldo Cruz,2017,112(4):281-291.
[24]Kulkarni P S,Sahai A,Gunale B,et al.Development of a new purified vero cell rabies vaccine(Rabivax-S)at the serum institute of India Pvt Ltd[J].Expert Rev Vaccines,2017,16(4):303-311.
[25]Qian Y,Jung Y S and Chen X.DEC1 and MIC-1:new players of p53-dependent cell fate decision[J].Cell Cycle,2012,11(19):3525-3526.
[26]Gossen M and Bujard H.Tight control of gene expression in mammalian cells by tetracycline-responsive promoters[J].Proc Natl Acad Sci,1992,89:5547-5551.
[27]Gossen M,Freundlieb S,Bender G,et al.Transcriptional activation by tetracyclines in mammalian cells[J].Science,1995,268:1766-1769.
[2]St-Pierre B,Flock G,Zacksenhaus E,et al.Stra13 homodimers repress transcription through class B E-box elements[J].J Biol Chem,2002,277(48):46544-46551.
[3]Qian Y J and Chen X.ID1,inhibitor of differentiation/DNA binding,is an effector of the p53-dependent DNA damage response pathway[J].J Biol Chem,2008,283(33):22410-22416.
[4]Qian Y J,Jung Y S and Chen X.Differentiated embryo-chondrocyte expressed gene 1 regulates p53-dependent cell survival versus cell death through macrophage inhibitory cytokine-1[J].Proc Natl Acad Sci U S A,2012,109(28):11300-11305.
[5]Li Y,Xie M,Yang J,et al.The expression of antiapoptotic protein survivin is transcriptionally upregulated by DEC1 primarily through multiple sp1 binding sites in the proximal promoter[J].Oncogene,2006,25(23):3296-3306.
[6]Qian Y J,Jung Y S,Chen X.Delta Np63,a target of DEC1 and histone deacetylase 2,modulates the efficacy of histone deacetylase inhibitors in growth suppression and keratinocyte differentiation[J].J Biol Chem,2011,286(14):12033-12041.
[7]Qian Y J,Zhang J,Jung Y S,et al.DEC1 coordinates with HDAC8to differentially regulate TAp73 and Delta Np73 expression[J].PLo S One,2014,9(1):e84015.
[8]Yamada K,Miyamoto K.Basic helix-loop-helix transcription factors,BHLHB2 and BHLHB3;their gene expressions are regulated by multiple extracellular stimuli[J].Front Biosci,2005,10:3151-3171.
[9]Fuyuki S,Ujjal K Bhawal,Tomohiro Yoshimura,et al.DEC1 and DEC2 crosstalk between circadian rhythm and tumor progression[J].J Cancer,2016,7(2):153-159.
[10]Sato F,Bhawal U K,Yoshimura T,et al.DEC1/STRA13/SHARP2and DEC2/SHARP1 coordinate physiological processes,including circadian rhythms in response to environmental stimuli[J].Curr Top Dev Biol,2014,110:339-372.
[11]Honma S,Kawamoto T,Takagi Y,et al.Dec1 and Dec2 are regulators of the mammalian molecular clock[J].Nature,2002,419(6909):841-844.
[12]Qian Y J,Zhang J,Yan B,et al.DEC1,a basic helix-loop-helix transcription factor and a novel target gene of the p53 family,mediates p53-dependent premature senescence[J].J Biol Chem,2008,283(5):2896-2905.
[13]Sun H,Lu B,Li R Q,et al.Defective T cell activation and autoimmune disorder in Stra13-deficient mice[J].Nat Immunol,2001,2(11):1040-1047.
[14]Miyazaki K,Miyazaki M,Guo Y,et al.The role of the basic helixloop-helix transcription factor Dec1 in the regulatory T cells[J].J Immunol,2010,185(12):7330-7339.
[15]Lin C C,Bradstreet T R,Schwarzkopf E A,et al.Bhlhe40 controls cytokine production by T cells and is essential for pathogenicity in autoimmune neuroinflammation[J].Nat Commun,2014,5:3551.
[16]Kanda M,Yamanaka H,Kojo S,et al.Transcriptional regulator Bhlhe40 works as a cofactor of T-bet in the regulation of IFNgamma production in i NKT cells[J].Proc Natl Acad Sci U S A,2016,113(24):E3394-3402.
[17]Bhawal U K.IL-1beta-mediated up-regulation of DEC1 in human gingiva cells via the Akt pathway[J].J Cell Biochem,2012,113(10):3246-3253.
[18]Ivanova A V,Ivanov S V,Zhang X,et al.STRA13 interacts with STAT3 and modulates transcription of STAT3-dependent targets[J].J Mol Biol,2004,340(4):641-653.
[19]Annamalai T,Saif L J,Lu Z,et al.Age-dependent variation in innate immune responses to porcine epidemic diarrhea virus infection in suckling versus weaned pigs[J].Vet Immunol Immunopathol,2015,168(3-4):193-202.
[20]Cecere T E,Todd S M and Leroith T.Regulatory T cells in arterivirus and coronavirus infections:do they protect against disease or enhance it[J].Viruses,2012,4(5):833-846.
[21]Charley B,Riffault S and Van Reeth K.Porcine innate and adaptative immune responses to influenza and coronavirus infections[J].Ann N Y Acad Sci,2006,1081:130-136.
[22]Nikolay A,Castilho L R,Reichl U,et al.Propagation of Brazilian zika virus strains in static and suspension cultures using vero and BHK cells[J].Vaccine,2017,S0264-410X(17):30324-30329.
[23]Castrillon-Betancur J C and Urcuqui-Inchima S.Overexpression of miR-484 and miR-744 in Vero cells alters dengue virus replication[J].Mem Inst Oswaldo Cruz,2017,112(4):281-291.
[24]Kulkarni P S,Sahai A,Gunale B,et al.Development of a new purified vero cell rabies vaccine(Rabivax-S)at the serum institute of India Pvt Ltd[J].Expert Rev Vaccines,2017,16(4):303-311.
[25]Qian Y,Jung Y S and Chen X.DEC1 and MIC-1:new players of p53-dependent cell fate decision[J].Cell Cycle,2012,11(19):3525-3526.
[26]Gossen M and Bujard H.Tight control of gene expression in mammalian cells by tetracycline-responsive promoters[J].Proc Natl Acad Sci,1992,89:5547-5551.
[27]Gossen M,Freundlieb S,Bender G,et al.Transcriptional activation by tetracyclines in mammalian cells[J].Science,1995,268:1766-1769.