分子诊断与治疗杂志
分子診斷與治療雜誌
분자진단여치료잡지
JOURNAL OF MOLECULAR DIAGNOSIS AND THERAPY
2015年
3期
161-165
,共5页
张玲%翁云层%张云%帅丽芳%李婷婷%王文敬%李红卫%赵卫%黎诚耀
張玲%翁雲層%張雲%帥麗芳%李婷婷%王文敬%李紅衛%趙衛%黎誠耀
장령%옹운층%장운%수려방%리정정%왕문경%리홍위%조위%려성요
重组慢病毒%绿色荧光蛋白
重組慢病毒%綠色熒光蛋白
중조만병독%록색형광단백
Recombinant lentivirus%Green florescent protein
目的:比较不同启动子的慢病毒转导细胞后,在不同细胞系中驱动绿色荧光蛋白表达的效率高低。方法采用3种不同启动子的转移质粒,与包装质粒共转染293T细胞,转染60 h后,收集慢病毒上清。用等量的三种慢病毒液转导5种细胞系(293A、MOLT-4、PC3、DU145及RM1),72 h后,荧光显微镜下观察转导效果;流式细胞仪计数转导效率。结果不同启动子( Ubiquitin , EF1α, CMV )在5种细胞系中驱动绿色荧光蛋白的表达及转导效率不同。在293A 和PC3细胞中,CMV 为最强启动子,转导率分别为(94.83±2.87)%和(20.90±3.15)%;但在MOLT-4和DU145细胞中,EF1α为最强启动子,转导率分别为(74.27±2.14)%和(25.13±4.95)%;在RM1细胞中,Ubiquitin为最强启动子,转导率为(16.77±0.38)%。
目的:比較不同啟動子的慢病毒轉導細胞後,在不同細胞繫中驅動綠色熒光蛋白錶達的效率高低。方法採用3種不同啟動子的轉移質粒,與包裝質粒共轉染293T細胞,轉染60 h後,收集慢病毒上清。用等量的三種慢病毒液轉導5種細胞繫(293A、MOLT-4、PC3、DU145及RM1),72 h後,熒光顯微鏡下觀察轉導效果;流式細胞儀計數轉導效率。結果不同啟動子( Ubiquitin , EF1α, CMV )在5種細胞繫中驅動綠色熒光蛋白的錶達及轉導效率不同。在293A 和PC3細胞中,CMV 為最彊啟動子,轉導率分彆為(94.83±2.87)%和(20.90±3.15)%;但在MOLT-4和DU145細胞中,EF1α為最彊啟動子,轉導率分彆為(74.27±2.14)%和(25.13±4.95)%;在RM1細胞中,Ubiquitin為最彊啟動子,轉導率為(16.77±0.38)%。
목적:비교불동계동자적만병독전도세포후,재불동세포계중구동록색형광단백표체적효솔고저。방법채용3충불동계동자적전이질립,여포장질립공전염293T세포,전염60 h후,수집만병독상청。용등량적삼충만병독액전도5충세포계(293A、MOLT-4、PC3、DU145급RM1),72 h후,형광현미경하관찰전도효과;류식세포의계수전도효솔。결과불동계동자( Ubiquitin , EF1α, CMV )재5충세포계중구동록색형광단백적표체급전도효솔불동。재293A 화PC3세포중,CMV 위최강계동자,전도솔분별위(94.83±2.87)%화(20.90±3.15)%;단재MOLT-4화DU145세포중,EF1α위최강계동자,전도솔분별위(74.27±2.14)%화(25.13±4.95)%;재RM1세포중,Ubiquitin위최강계동자,전도솔위(16.77±0.38)%。
Objective To compare the GFP expression efficiency of transduced cells driven by lenti-virus with different promoters. Methods 293T cells were co-transfected with packaging plasmids and three kinds of transfer plasmids. Then the supernatant was collected after 60 hours. 5 cell lines (293A, MOLT-4, PC3, DU145 and RM1) were transduced by equal load of these lentivirus. 72 hours later, GFP expression was ob-served with florescence microscope and transduction efficency was counted by FASC. Results GFP expres-sion and transduction efficiency among 5 cell lines were different. Among 293A and PC3 cells, CMV promoter was the strongest one and transduction efficiency can achieve (94.83 ± 2.87)% and (20.90 ± 3.15)%, respec-tively. But among MOLT-4 and DU145 cells, EF1α promoter was the strongest one and transduction efficiency can achieve (74.27 ± 2.14)%and (25.13 ± 4.95)%, respectively. In RM1 cells, Ubiquitin promoter was the strongest one and it can achieve (16.77 ± 0.38)%. Conclusion In the study of gene expression mediated by lentivirus, proper cell lines and promoters should be selected to obtain high efficiency.