山东医药
山東醫藥
산동의약
SHANDONG MEDICAL JOURNAL
2014年
42期
4-7
,共4页
Six2%胶质细胞系源性神经营养因子%慢病毒%载体构建%基因敲减
Six2%膠質細胞繫源性神經營養因子%慢病毒%載體構建%基因敲減
Six2%효질세포계원성신경영양인자%만병독%재체구건%기인고감
Six2%glial cellline-derived neurotrophic factor%lentiviral%vector construction%gene kncokdown
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pLV-shSix2-1、pLV-shSix2-2、pLV-shSix2-3）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pLV-H1-EF1a载体质粒，均采用慢病毒包装四质粒系统（pRRE/pVSVG/pREV/pLV）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 pLV-shSix2。收集敲减Six2基因的pLV-shSix2-1、pLV-shSix2-2、pLV-shSix2-3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pLV-shSix2-1组、pLV-shSix2-2组、pLV-shSix2-3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9000 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shSix2-2对应的细胞中Six2蛋白表达显著降低，shSix2-1和shSix2-3蛋白表达降低效果不明显。用嘌呤霉素筛选病毒感染的pLV-shSix2-2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。
目的：篩選併包裝Six2基因shRNA敲減的慢病毒，建立穩定錶達敲減Six2基因的黑質多巴胺能神經元MES23．5細胞繫。方法閤成三種（分彆命名為pLV-shSix2-1、pLV-shSix2-2、pLV-shSix2-3）含敲減Six2基因榦擾序列的雙鏈DNA oligo，併將其連接于含有嘌呤黴素抗性的pLV-H1-EF1a載體質粒，均採用慢病毒包裝四質粒繫統（pRRE/pVSVG/pREV/pLV）包裝併轉染腺病毒 E1A 基因的人腎上皮細胞繫（293T 細胞）以穫取慢病毒 pLV-shSix2。收集敲減Six2基因的pLV-shSix2-1、pLV-shSix2-2、pLV-shSix2-3慢病毒質粒及對照質粒上清，感染黑質多巴胺能神經元細胞（MES23．5細胞），分彆設為pLV-shSix2-1組、pLV-shSix2-2組、pLV-shSix2-3組及對照組，Western blot法檢測各組細胞 Six2蛋白錶達，鑒定各組Six2榦擾序列的敲減效果。嘌呤黴素法篩選穩定敲減 Six2的MES23．5細胞株。結果酶切結果錶明在262 bp及約9000 bp處分彆有明顯條帶，測序結果顯示所測序列與敲減的Six2榦擾序列完全一緻。 Western blot結果顯示榦擾序列shSix2-2對應的細胞中Six2蛋白錶達顯著降低，shSix2-1和shSix2-3蛋白錶達降低效果不明顯。用嘌呤黴素篩選病毒感染的pLV-shSix2-2組細胞繫成功。結論成功構建併篩選瞭Six2基因shRNA敲減慢病毒錶達載體，建立瞭穩定敲減Six2基因的MES23．5細胞株。
목적：사선병포장Six2기인shRNA고감적만병독，건립은정표체고감Six2기인적흑질다파알능신경원MES23．5세포계。방법합성삼충（분별명명위pLV-shSix2-1、pLV-shSix2-2、pLV-shSix2-3）함고감Six2기인간우서렬적쌍련DNA oligo，병장기련접우함유표령매소항성적pLV-H1-EF1a재체질립，균채용만병독포장사질립계통（pRRE/pVSVG/pREV/pLV）포장병전염선병독 E1A 기인적인신상피세포계（293T 세포）이획취만병독 pLV-shSix2。수집고감Six2기인적pLV-shSix2-1、pLV-shSix2-2、pLV-shSix2-3만병독질립급대조질립상청，감염흑질다파알능신경원세포（MES23．5세포），분별설위pLV-shSix2-1조、pLV-shSix2-2조、pLV-shSix2-3조급대조조，Western blot법검측각조세포 Six2단백표체，감정각조Six2간우서렬적고감효과。표령매소법사선은정고감 Six2적MES23．5세포주。결과매절결과표명재262 bp급약9000 bp처분별유명현조대，측서결과현시소측서렬여고감적Six2간우서렬완전일치。 Western blot결과현시간우서렬shSix2-2대응적세포중Six2단백표체현저강저，shSix2-1화shSix2-3단백표체강저효과불명현。용표령매소사선병독감염적pLV-shSix2-2조세포계성공。결론성공구건병사선료Six2기인shRNA고감만병독표체재체，건립료은정고감Six2기인적MES23．5세포주。
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.
