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目的构建SATB1基因慢病毒载体,为其体内外实验研究提供依据。方法将靶向SATB1基因的shRNA表达序列连接到慢病毒载体pGCSIL-GFP中,获得pGCSIL-GFP-shSATB1质粒,经PCR和测序鉴定后与慢病毒包装质粒pGCSIL-GFP-shSATB1通过Lipofectamine2000共转染至细胞293T,包装产生病毒液,测定其滴度。结果PCR扩增和测序结果证实,SATB1shRNA核苷酸链序列插入正确,包装慢病毒产生病毒悬液的滴度为2×107pfu/ml。结论成功构建人SATB1基因慢病毒载体。
Objective To construct lentiviral vector of SATB1 gene and provide basis for its in vitro and in vivo experiments. Methods The shRNA expression sequence targeting SATB1 gene was ligated into the lentiviral vector pGCSIL-GFP to obtain the pGCSIL-GFP-shSATB1 plasmid. The recombinant plasmid pGCSIL-GFP-shSATB1 was co-transfected with the lentivirus packaging plasmid pGCSIL-GFP-shSATB1 by Lipofectamine 2000 Cells 293T, packaging virus liquid, measured its titer. Results The results of PCR amplification and sequencing confirmed that the sequence of SATB1 shRNA nucleotide insert was inserted correctly and the titer of the lentivirus-producing virus suspension was 2 × 107pfu / ml. Conclusion The human SATB1 gene lentiviral vector was successfully constructed.