PLEKHQ1基因敲除小鼠的永生化骨髓来源巨噬细胞系的建立
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摘要
目的:建立PLEKHQ1基因敲除小鼠的永生化骨髓来源巨噬细胞系,为PLEKHQ1基因功能的研究奠定基础。方法:用慢病毒感染的方法将包装质粒pCDH-SV40/GFP导入野生型和PLEKHQ1基因敲除小鼠的骨髓来源巨噬细胞,建立永生化细胞系;观察永生化细胞的生物学性状,用聚合酶链反应(PCR)检测目的基因的整合,用RT-PCR鉴定目的基因的表达,并对永生化和非永生化骨髓来源巨噬细胞的生长状况进行比较。结果:构建的骨髓来源巨噬细胞系已扩大培养并稳定传代,经鉴定,SV40LT抗原已整合入细胞并稳定表达。结论:通过慢病毒感染细胞的方法成功构建了PLEKHQ1基因敲除小鼠的永生化骨髓来源巨噬细胞系。
Objective: To establish an immortalized bone marrow-derived macrophage(BMDM) cell line in PLEKHQ1 knockout mouse so as to pave the way for further studies on the function of PLEKHQ1 gene. Methods: Packaging plasmid pCDH-SV40/GFP was transfected into wild type mouse BMDM and PLEKHQ1 knockout mouse BMDM by the means of lentivirus infection so as to establish immortalized cell lines. The cell morphology of immortalized cell lines was observed and Polymerase chain reaction(PCR)was used to detect the integration of the target gene, and RTPCR was used to identified the expression of target gene. The proliferation between the immortalized BMDM and nonimmortalized BMDM was compared and analyzed in the finally. Results: The established immortalized BMDM cell line has achieved stable growth and serial propagation, and the results of identification showed that SV40 LT antigen gene has been integrated in cell and been stably expressed. Conclusion: An immortalized BMDM cell line of PLEKHQ1 knockout mouse has been successfully established by the mean of. lentivirus infecting cells.
Objective: To establish an immortalized bone marrow-derived macrophage(BMDM) cell line in PLEKHQ1 knockout mouse so as to pave the way for further studies on the function of PLEKHQ1 gene. Methods: Packaging plasmid pCDH-SV40/GFP was transfected into wild type mouse BMDM and PLEKHQ1 knockout mouse BMDM by the means of lentivirus infection so as to establish immortalized cell lines. The cell morphology of immortalized cell lines was observed and Polymerase chain reaction(PCR)was used to detect the integration of the target gene, and RTPCR was used to identified the expression of target gene. The proliferation between the immortalized BMDM and nonimmortalized BMDM was compared and analyzed in the finally. Results: The established immortalized BMDM cell line has achieved stable growth and serial propagation, and the results of identification showed that SV40 LT antigen gene has been integrated in cell and been stably expressed. Conclusion: An immortalized BMDM cell line of PLEKHQ1 knockout mouse has been successfully established by the mean of. lentivirus infecting cells.
引文
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[8]李培杰,赵国强.重组SV40大T抗原慢病毒表达载体的构建.肿瘤基础与临床,2008,21(5):384-387.
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[12]Inozemtseva LS,Chemikov VG,Manuilova ES,et al.Immortalization of human fibroblasts using ts A mutant of SV40 and p SV3neo plasmid[J].Ts itologiia,2001,43(10):944-953.
[13]Markovics JA,Carroll PA,Robles MT,et al.Intestinal dysplasia induced by simian virus 40 T antigen is independent of p53[J].J Virol,2005,79(12):7492-7502.
[2]Boch J,Scholze H,Schornack S,et al.Breakingthe code of DNA binding specificity of TAL-typeⅢeffectors[J].Science,2009,326(5959):1509-1512.
[3]Li T,Huang S,Jiang WZ,et al.TAL nucleases(TALNs):hybrid proteins composed of TAL effectors and FokⅠDNA-cleavage domain[J].Nucleic Acids Res,2011,39(1):359-372.
[4]Kim Y,Kweon J,Kim A,et al.A library of TAL effector nucleases spanning the human genome[J].Nat Biotechnol,2013,31(3):251-258.
[5]Moscou MJ,Bogdanove AJ.A simple cipher governs DNA recognition by TAL effectors[J].Science,2009,326(5959):1501-1503.
[6]Gingras AC,Raught B,Sonenberg N.el F4initiation factors:effectors of m RNA recruitment to ribosomes and regulators of translation[J].Annu Rev Biochem,1999(68):913-963.
[7]张鹏飞,张硌,陆琤,等.PLEKHQ1基因敲除小鼠基因型鉴定方法[J].中国医学装备,2015,12(8):1-3.
[8]李培杰,赵国强.重组SV40大T抗原慢病毒表达载体的构建.肿瘤基础与临床,2008,21(5):384-387.
[9]Obinata M.Immortalized cell lines with differentiation potentials:their establishment and possible application[J].Seikagaku,2008,80(1):5-12.
[10]Yu Y,Alwine JC.Interaction between simian virus 40 large T antigen and insulin receptor substrate1 is disrupted by the K1mutation,resulting in the loss of large T antigen-mediated phosphorylation of Akt[J].J Virol,2008,82(9):4521-4526.
[11]Kirchhoff C,Araki Y,Huhtaniemi I,et al.Immortalization by large T-antigen of the adult epididymal duct epithelium[J].Mol Cell Endocrinol,2004,216(1/2):83-94.
[12]Inozemtseva LS,Chemikov VG,Manuilova ES,et al.Immortalization of human fibroblasts using ts A mutant of SV40 and p SV3neo plasmid[J].Ts itologiia,2001,43(10):944-953.
[13]Markovics JA,Carroll PA,Robles MT,et al.Intestinal dysplasia induced by simian virus 40 T antigen is independent of p53[J].J Virol,2005,79(12):7492-7502.