【作者】 刘中华；

【导师】 王华岩；

【作者基本信息】 西北农林科技大学 ， 动物生物技术， 2011， 博士

【摘要】 本研究以增强型绿色荧光蛋白基因(EGFP)为靶标,分别设计构建茎部长度为21 bp、27 bp和29 bp的shRNA表达载体,通过转染细胞和显微注射对不同茎部长度的shRNA表达载体在小鼠细胞及个体水平的干扰效应做一系统的评估并优化条件,以期建立、提高并完善小鼠基因沉默技术体系,为小鼠个体水平的RNA干扰研究提供基础性数据和资料。1.从13.5 d的绿色荧光小鼠胎儿中分离培养成纤维细胞,胰蛋白酶消化法处理,经3~4 d培养,细胞即可铺满瓶底,比组织块法(6~8 d)所需时间短。细胞传代时,0.25%胰蛋白酶+0.02% EDTA消化耗时最短,细胞贴壁率较高。试验表明绿色荧光小鼠胎儿成纤维细胞最适合的培养液为DMEM(高糖)+10% FBS,并确定了其最佳G418筛选浓度为400μg/mL。2.以EGFP为干扰靶基因,设计茎部长度为21 bp、27 bp和29 bp的shRNA序列,然后分别连入psiSTRIKE表达载体中,酶切及测序结果证明shRNA序列成功的克隆进入表达载体中。3.将构建好的载体一部分借助脂质体转染小鼠胎儿成纤维细胞,另一部分直接注射入小鼠腿部肌肉,利用实时荧光定量RT-PCR对其荧光表达进行精确定量。细胞水平上,21 bp、27 bp和29 bp茎部长度shRNA表达载体分别使得目的基因表达降低到45%、32%、25%;21 bp最大沉默效应出现在转染后24 h,而27 bp和29 bp出现在48 h。肌肉注射使得靶基因的表达水平分别下降到原来的8.9%、9.7%、9.0%,不同茎部载体沉默效应无显著差异。4.以EGFP为干扰靶基因,分别构建了具有荧光标记的茎部长度为21 bp、27 bp、29 bp的一系列串联shRNA干扰载体。将构建的干扰载体利用脂质体法分别转染Vero细胞,荧光显微镜下观察EGFP表达变化,仅见少量细胞发出微弱荧光。利用实时荧光定量RT-PCR对不同转染组沉默效应进行定量分析,结果表明pGenesilR21、pGenesilR27和pGenesilR29荧光表达量仅为对照组(pEGFP-C1)的4.3%、3.0%和6.9%。5.构建的不同茎部长度串联shRNA干扰载体分别注射小鼠腿部肌肉,实时荧光定量RT-PCR对其沉默效应进行定量分析,结果表明pGenesilR21、pGenesilR27和pGenesilR29荧光表达量仅为对照质粒(pEGFP-C1)的2.5%、1.3%和5.1%。6.小鼠胚胎原核显微注射干扰载体后进行体外培养,发育多数阻滞在二细胞期,此时荧光显微镜下可观察到明显的荧光减弱现象。本试验共注射受精卵1192枚,注射后存活763枚,存活率64%。在移植的44只母鼠中,仅有23只怀孕,中途流产21只,最终顺利产仔一窝。7.通过尾静脉注射将构建的干扰载体导入小鼠体内以初步评价其负效应,小鼠在注射后两个月内,均未见死亡及其它不良反应。PCR检测发现试验小鼠体内有目标基因的整合。8.转基因小鼠后代经PCR检测,共有5个阳性。更多还原

【Abstract】 In this study, shRNA expression vectors with different stem length (21 bp, 27 bp, and 29 bp) were constructed, and the enhanced green fluorescent protein gene was used as target gene. Their silencing effect was tested in mouse cell and in vivo by cell transfection and pronuclear microinjection. The findings in this study are of interest for selecting the best hairpins for mouse individuals.1. Mouse embryonic fibroblasts were isolated and cultured from EGFP-transgenic mouse. Cells were more profuse and pure acquired from13.5 days embryonic age than other ages. It took about 3-4 days that the cell spreaded out the bottom of culture flask by trypsin digestion method, which was shorter than that (6-8 days) by tissue block method. Compared with other methods, it is time saving and has a high attachment rate that digested by 0.25% trypsin+ 0.02% EDTA for this cell passage. After several rounds of screening, DMEM(high glucose)+10% FBS was deemed to be the optimal culture medium, and 400μg/mL was considered the suitable selection concentration of G418 for EGFP -transgenic mouse embryonic fibroblasts.2. Aiming directly at the enhanced green fluorescent protein gene, shRNAs with different stem length (21 bp, 27 bp, and 29 bp) were designed and then ligated into psiSTRIKE expression vector. Target sequences were successfully cloned into the vector testified by enzyme digestion and sequencing.3. The recombinant plasmids were transfected into mouse embryonic fibroblast with lipofection and injected into leg muscle of mouse separately. The mRNA expression level of green fluorescent protein gene was checked by real-time fluorescent quantitative RT-PCR. EGFP-21siRNA, EGFP-27siRNA and EGFP-29siRNA regulated the gene expression down to 45%, 32%, 25%, respectively in cell. The silencing effect reached peak at 24h after transfection by EGFP-21siRNA and at 48h by EGFP-27siRNA or EGFP-29siRNA. EGFP-21siRNA, EGFP-27siRNA and EGFP-29 siRNA regulated the expression of target gene down to 8.9%, 9.7%, and 9.0%, respectively in vivo. There were no obvious difference among silencing effect of shRNAs with stem length of 21 bp, 27 bp and 29 bp. 4. Fluorescence labeled vectors of tandem shRNA with different stem length (21 bp, 27 bp, and 29 bp) targeting EGFP were successfully constructed. There was only light fluorescence observed under fluorescence microscope when these vectors were transfected into Vero cells. The mRNA expression level of green fluorescent protein gene was checked by real-time fluorescent quantitative RT-PCR. The results showed that the fluorescent expression of pGenesilR21, pGenesilR27, and pGenesilR29 was only 4.3%, 3.0% and 6.9% of the control group (pEGFP-C1) separately.5. The tandem shRNA expression vectors were injected into leg muscle of mouse. The mRNA expression level of green fluorescent protein gene was checked by real-time fluorescent quantitative RT-PCR. The results showed that fluorescent expression of pGenesilR21, pGenesilR27, and pGenesilR29 was only 2.5%, 1.3% and 5.1% of the control group (pEGFP-C1) separately.6. The interfering vectors were microinjected into male pronucleus of the embryos. When cultured in vitro, embryo developmental arrest took place in 2 cell stage. It was obvious that the fluorescence of microinjected embryos became weakened under microscope. 1192 of mouse fertilized eggs were microinjected altogether, and 763 of them survived after treatment in this study. Survival rate was about 64%. Only 23 of 44 transplanted female mice had pregnancies. There were 21 of mother mice aborting and only one giving birth to living offspring in the gestation period.7. The interfering vectors were injected into mouse tail vein in order to value their adverse effect. All of the mice did not die and had no adverse effect two months later after injection, while target gene had integrated in the mouse genome testified by PCR.8. 5 samples were positive in progeny of transgenic mouse detected by PCR method.更多还原