Construction of Six2 shRNA lentiviral vector and kncokdown Six2 stably cell line

GAO Jin; KANG Xiao-yu

doi:10.3969/j.issn.1002-266X.2014.42.002

Abstract：Objective To screen and pack the Six2 shRNA knockdown lentiviral and to establish the Six2 stable knockdown MES23 .5 cell line.Methods We synthesized 3 different double-strand DNA oligos containing interfer-ence sequence of Six2 ( each sequence named as pLV-shSix2-1 , pLV-shSix2-2 and pLV-shSix2-3 ) and linked these genes to pLV-H1-EF1 a vector which contains puromycin resistance.Then we transfected and packed these lentiviral packaging systems (pRRE/pVSVS/pREV/pLV) into adenovirus E1A gene renal epithelial cell line (293T) in order to obtain the pLV-shSix2 letivirus.Furthermore, we collected the supernatant of the 3 knockdown plasmids of Six2 and the control plasmid to infect the domaninergic neurons in substantia nigra (MES23.5 cells).Cells were divided into pLV-shSix2-1 group, pLV-shSix2-2 group, pLV-shSix2-3 group and control group.Western blot analysis was used to detected the expression levels of six2 and identify the knockdown effects of Six2 .Puromycin method was used to screen the cell lines stably knockdown Six2 expression.Results Results of digestion using restriction enzyme and agarose gel electrophoresis showed that there were two obvious bands at 260 bp and 9 000 bp.Sequencing results showed that the interference sequencing of six2 was exactly correct.There was nearly no cell death at the fifth generation after the screening of puromycin, and the Six2 protein expression were greatly decreased in cells expressing shSix2-2 sequence, however, the expression of Six2 did not changed in cells expressing the shSix2-1 or shSix2-3 sequence.Conclusions We successfully constructed the Six2 knockdown lentiviral vector and established the MES23 .5 cell line stably ex-pressing Six2 interference sequence.