中文摘要：

目的筛选并包装Six2基因shRNA敲减的慢病毒,建立稳定表达敲减Six2基因的黑质多巴胺能神经元MES23.5细胞系。方法合成三种（分别命名为p LV-sh Six2-1、p LV-sh Six2-2、p LV-sh Six2-3）含敲减Six2基因干扰序列的双链DNA oligo,并将其连接于含有嘌呤霉素抗性的p LV-H1-EF1a载体质粒,均采用慢病毒包装四质粒系统（pRRE/p VSVG/pREV/p LV）包装并转染腺病毒E1A基因的人肾上皮细胞系（293T细胞）以获取慢病毒p LVsh Six2。收集敲减Six2基因的p LV-sh Six2-1、p LV-sh Six2-2、p LV-sh Six2-3慢病毒质粒及对照质粒上清,感染黑质多巴胺能神经元细胞（MES23.5细胞）,分别设为p LV-sh Six2-1组、p LV-sh Six2-2组、p LV-sh Six2-3组及对照组,Western blot法检测各组细胞Six2蛋白表达,鉴定各组Six2干扰序列的敲减效果。嘌呤霉素法筛选稳定敲减Six2的MES23.5细胞株。结果酶切结果表明在262 bp及约9 000 bp处分别有明显条带,测序结果显示所测序列与敲减的Six2干扰序列完全一致。Western blot结果显示干扰序列sh Six2-2对应的细胞中Six2蛋白表达显著降低,sh Six2-1和sh Six2-3蛋白表达降低效果不明显。用嘌呤霉素筛选病毒感染的p LV-sh Six2-2组细胞系成功。结论成功构建并筛选了Six2基因shRNA敲减慢病毒表达载体,建立了稳定敲减Six2基因的MES23.5细胞株。

英文摘要：

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 interference sequence of Six2（ each sequence named as p LV-sh Six2-1,p LV-sh Six2-2 and p LV-sh Six2-3） and linked these genes to p LV-H1-EF1 a vector which contains puromycin resistance. Then we transfected and packed these lentiviral packaging systems（ pRRE / p VSVS / pREV / p LV） into adenovirus E1 A gene renal epithelial cell line（ 293 T） in order to obtain the p LV-sh Six2 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 p LV-sh Six2-1 group,p LV-sh Six2-2 group,p LV-sh Six2-3 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 sh Six2-2 sequence,however,the expression of Six2 did not changed in cells expressing the sh Six2-1 or sh Six2-3 sequence. Conclusions We successfully constructed the Six2 knockdown lentiviral vector and established the MES23. 5 cell line stably expressing Six2 interference sequence.