慢病毒介导的miR194-5p过表达精原细胞株的建立
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摘要
目的构建miR194-5p慢病毒表达载体pMSCV-PIG-miR194-5p,应用包装后产生的慢病毒感染Stra8-GC1-spg细胞,获得稳定过表达miR194-5p精原细胞株。方法用PCR法扩增miR194-5p的前体序列pri-miR194-5p。利用分子克隆技术构建慢病毒表达载体pMSCV-PIG-miR194-5p。将其与Gag pol和VSV.G辅助质粒一起用PEI法共转染至293T包装细胞内,收集慢病毒上清,后将其感染Stra8-GC1-spg细胞,经嘌呤霉素筛选获得稳定过表达miR194-5p的精原细胞株。结果构建了慢病毒重组质粒pMSCV-PIG-miR194-5p。经酶切鉴定及DNA测序法证实序列准确无误。经包装产生的慢病毒能成功感染Stra8-GC1-spg细胞,并稳定过表达miR194-5p。结论成功地构建了慢病毒表达重组质粒pMSCV-PIG-miR194-5p,经包装后产生预计的慢病毒,建立了稳定过表达miR194-5p的精原细胞株。
Objective To construct a recombinant lentivirus expression vector pMSCV-PIG-miR194-5p, then obtain LV-miR194-5p-OE by packaging in 293T cell, ultimately establish stabling spermatogonial cells(Stra8-GC1 cells) with miR194-5p overexpression by transfection. Methods The pri-miR194-5p fragment was amplified by PCR. Recombinant expression plasmid pMSCV-PIG-miR194-5p was firstly constructed by molecular cloning technology, then the recombinant expression plasmid was identified by double digestion and sequence analysis. The recombinant plasmid and assisting plasmid Gag pol and VSV.G were co-transfected into 293T cell by PEI and their supernatant were harvested after 48 hours and 72 hours culture. Stra8-GC1 cells were infected with the collected lentivirus particle in the supernatant. Spermatogonial cells with stability miR194-5p over-expressing were established by puromycin selection for one week. Results Recombinant expression plasmid pMSCV-PIG-miR194-5p were successfully confirmed by enzyme digestion and DNA sequence analysis. Stra8-GC1-spg cells with stability miR194-5p overexpression were successfully established by transfection with LVpMSCV-PIG-miR194-5p. Conclusion Construct lentivirus recombinant plasmid pMSCV-PIG-miR194-5p successfully, producing the lentivirus after packaging in 293T cells, acquiring over-expressing miR194-5p stability Stra8-GC1-spg cells.
Objective To construct a recombinant lentivirus expression vector pMSCV-PIG-miR194-5p, then obtain LV-miR194-5p-OE by packaging in 293T cell, ultimately establish stabling spermatogonial cells(Stra8-GC1 cells) with miR194-5p overexpression by transfection. Methods The pri-miR194-5p fragment was amplified by PCR. Recombinant expression plasmid pMSCV-PIG-miR194-5p was firstly constructed by molecular cloning technology, then the recombinant expression plasmid was identified by double digestion and sequence analysis. The recombinant plasmid and assisting plasmid Gag pol and VSV.G were co-transfected into 293T cell by PEI and their supernatant were harvested after 48 hours and 72 hours culture. Stra8-GC1 cells were infected with the collected lentivirus particle in the supernatant. Spermatogonial cells with stability miR194-5p over-expressing were established by puromycin selection for one week. Results Recombinant expression plasmid pMSCV-PIG-miR194-5p were successfully confirmed by enzyme digestion and DNA sequence analysis. Stra8-GC1-spg cells with stability miR194-5p overexpression were successfully established by transfection with LVpMSCV-PIG-miR194-5p. Conclusion Construct lentivirus recombinant plasmid pMSCV-PIG-miR194-5p successfully, producing the lentivirus after packaging in 293T cells, acquiring over-expressing miR194-5p stability Stra8-GC1-spg cells.
引文
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14 Yu M,Mu H,Niu Z,et al.mi R-34c enhances mouse spermatogonial stem cells differentiation by targeting Nanos2.J Cell Biochem 2014;115(2):232-242
15 Wang Y,Zuo Q,Bi Y,et al.miR-31 Regulates Spermatogonial Stem Cells Meiosis via Targeting Stra8.J Cell Biochem 2017;118(12):4844-4853
16 Wang SH,Wu XC,Zhang MD,et al.Long noncoding RNA H19 contributes to gallbladder cancer cell proliferation by modulated mi R-194-5p targeting AKT2.Tumour Biol 2016;37(7):9721-9730
17 Huo D,Clayton WM,Yoshimatsu TF,et al.Identification of a circulating micro RNA signature to distinguish recurrence in breast cancer patients.Oncotarget 2016;7(34):55231-55248
18 Lewinska A,Adamczyk-Grochala J,Kwasniewicz E,et al.Reduced levels of methyltransferase DNMT2 sensitize human fibroblasts to oxidative stress and DNA damage that is accompanied by changes in proliferation-related mi RNA expression.Redox Biol 2018;14:20-34
19 van der Ree MH,Jansen L,Kruize Z,et al.Plasma MicroRNA Levels Are Associated With Hepatitis B e Antigen Status and Treatment Response in Chronic Hepatitis B Patients.J Infect Dis 2017;215(9):1421-1429
20 Deng R,Yue Y,Jin F,et al.Revisit the complexation of PEI and DNA-How to make low cytotoxic and highly efficient PEI gene transfection non-viral vectors with a controllable chain length and structure?J Control Release2009;140(1):40-46
21 Cryan SA,O'Driscoll CM.Mechanistic studies on nonviral gene delivery to the intestine using in vitro differentiated cell culture models and an in vivo rat intestinal loop.Pharm Res 2003;20(4):569-575
22 Kichler A,Leborgne C,Coeytaux E,et al.Polyethylenimine-mediated gene delivery:a mechanistic study.J Gene Med 2001;3(2):135-144
23 Brunner S,Sauser T,Carotta S,et al.Cell cycle dependence of gene transfer by lipoplex,polyplex and recombinant adenovirus.Gene Ther 2000;7(5):401-407
24 Lin CW,Jan MS,Kuo JS.The vector-related influences of autophagic micro RNA delivery by Lipofectamine 2000and polyethylenimine 25K on mouse embryonic fibroblast cells.Eur J Pharm Sci 2017;101:11-21
2 Yang Q,Hua J,Wang L,et al.MicroRNA and pi RNA profiles in normal human testis detected by next generation sequencing.PLo S One 2013;8(6):e66809
3 Rahbar S,Novin MG,Alizadeh E,et al.New insights into the expression profile of MicroRNA-34c and P53 in infertile men spermatozoa and testicular tissue.Cell Mol Biol(Noisy-le-grand)2017;63(8):77-83
4 Boissière A,Gala A,Ferrières-Hoa A,et al.Cell-free and intracellular nucleic acids:new non-invasive biomarkers to explore male infertility.Basic Clin Androl 2017;27:7
5 Kotaja N.MicroRNAs and spermatogenesis.Fertil Steril2014;101(6):1552-1562
6 Lian J,Zhang X,Tian H,et al.Altered micro RNA expression in patients with non-obstructive azoospermia.Reprod Biol Endocrinol 2009;7:13
7 Abu-Halima M,Backes C,Leidinger P,et al.MicroRNA expression profiles in human testicular tissues of infertile men with different histopathologic patterns.Fertil Steril2014;101(1):78-86.e2
8 Smorag L,Zheng Y,Nolte J,et al.MicroRNA signature in various cell types of mouse spermatogenesis:evidence for stage-specifically expressed mi RNA-221,-203and-34b-5p mediated spermatogenesis regulation.Biol Cell 2012;104(11):677-692
9 Bouhallier F,Allioli N,Lavial F,et al.Role of miR-34c micro RNA in the late steps of spermatogenesis.RNA2010;16(4):720-731
10 Bao J,Li D,Wang L,et al.MicroRNA-449 and microRNA-34b/c function redundantly in murine testes by targeting E2F transcription factor-retinoblastoma protein(E2F-p Rb)pathway.J Biol Chem 2012;287(26):21686-21698
11 Huszar J M,Payne CJ.MicroRNA 146(Mir146)modulates spermatogonial differentiation by retinoic acid in mice.Biol Reprod 2013;88(1):15
12 Yang QE,Racicot KE,Kaucher AV,et al.MicroRNAs221and 222 regulate the undifferentiated state in mammalian male germ cells.Development 2013;140(2):280-290
13 Mark M,Jacobs H,Oulad-Abdelghani M,et al.STRA8-deficient spermatocytes initiate,but fail to complete,meiosis and undergo premature chromosome condensation.J Cell Sci 2008;121(Pt 19):3233-3242
14 Yu M,Mu H,Niu Z,et al.mi R-34c enhances mouse spermatogonial stem cells differentiation by targeting Nanos2.J Cell Biochem 2014;115(2):232-242
15 Wang Y,Zuo Q,Bi Y,et al.miR-31 Regulates Spermatogonial Stem Cells Meiosis via Targeting Stra8.J Cell Biochem 2017;118(12):4844-4853
16 Wang SH,Wu XC,Zhang MD,et al.Long noncoding RNA H19 contributes to gallbladder cancer cell proliferation by modulated mi R-194-5p targeting AKT2.Tumour Biol 2016;37(7):9721-9730
17 Huo D,Clayton WM,Yoshimatsu TF,et al.Identification of a circulating micro RNA signature to distinguish recurrence in breast cancer patients.Oncotarget 2016;7(34):55231-55248
18 Lewinska A,Adamczyk-Grochala J,Kwasniewicz E,et al.Reduced levels of methyltransferase DNMT2 sensitize human fibroblasts to oxidative stress and DNA damage that is accompanied by changes in proliferation-related mi RNA expression.Redox Biol 2018;14:20-34
19 van der Ree MH,Jansen L,Kruize Z,et al.Plasma MicroRNA Levels Are Associated With Hepatitis B e Antigen Status and Treatment Response in Chronic Hepatitis B Patients.J Infect Dis 2017;215(9):1421-1429
20 Deng R,Yue Y,Jin F,et al.Revisit the complexation of PEI and DNA-How to make low cytotoxic and highly efficient PEI gene transfection non-viral vectors with a controllable chain length and structure?J Control Release2009;140(1):40-46
21 Cryan SA,O'Driscoll CM.Mechanistic studies on nonviral gene delivery to the intestine using in vitro differentiated cell culture models and an in vivo rat intestinal loop.Pharm Res 2003;20(4):569-575
22 Kichler A,Leborgne C,Coeytaux E,et al.Polyethylenimine-mediated gene delivery:a mechanistic study.J Gene Med 2001;3(2):135-144
23 Brunner S,Sauser T,Carotta S,et al.Cell cycle dependence of gene transfer by lipoplex,polyplex and recombinant adenovirus.Gene Ther 2000;7(5):401-407
24 Lin CW,Jan MS,Kuo JS.The vector-related influences of autophagic micro RNA delivery by Lipofectamine 2000and polyethylenimine 25K on mouse embryonic fibroblast cells.Eur J Pharm Sci 2017;101:11-21