转座子介导细胞质显微注射技术的基因转移效率比较

发布时间：2018-12-24 07:13

【摘要】：为了比较和探索SB(sleeping beauty)、PB(piggy Bac)和Tol2 3种转座子介导下的细胞质显微注射法在小鼠(Mus musculus)和斑马鱼(Danio rerio)上的转基因效率,本研究将包含3种转座子的重组载体p T3-PST-CAG-GFP分别与3种转座酶表达载体以1∶1质量比混合,显微注射至小鼠和斑马鱼受精卵细胞质,经体外培养后在胚胎发育不同时期用荧光显微镜检测报告基因绿色荧光蛋白(green fluorescent protein,GFP)阳性率,结果显示,在培养至4 Ed(96 h)(Ed:embryo day,胚胎发育的天数)的小鼠胚胎中,PB转座子介导的GFP转基因阳性率最高,达52%(N=281),显著高于SB的47%(N=328)和Tol2的36%(N=273)(P0.05);而受精后36和60 h(hour past fertilization,hpf)斑马鱼胚胎中,荧光显微镜检测表明,Tol2的GFP阳性率分别为36%(N=677)和37%(N=685),显著高于SB(28%N=691和27%N=708)和PB(30%N=687和32%N=675)(P0.05);转座子介导的细胞质显微注射可获得较高的转基因效率,且转座子活性具有物种差异性,在哺乳动物胚胎中PB转座子的基因转移效率最高,而在鱼类胚胎中Tol2转座子的基因转移效率最高。本研究为提高动物转基因效率研究提供重要参考资料。
[Abstract]:To compare and explore the transgene efficiency of three transposon-mediated cytoplasmic microinjection methods (SB (sleeping beauty), PB (piggy Bac) and Tol2) on mouse (Mus musculus) and zebrafish (Danio rerio). In this study, the recombinant vector p T3-PST-CAG-GFP containing three transposons was mixed with three transposase expression vectors at 1:1 mass ratio and microinjected into the zygote cytoplasm of mouse and zebrafish fertilized eggs. The positive rate of green fluorescent protein (green fluorescent protein,GFP) was detected by fluorescence microscope at different stages of embryonic development after in vitro culture. The results showed that the positive rate of green fluorescent protein (GFP) was 4 Ed (96 h) (Ed:embryo day,) in vitro. The positive rate of PB transposon mediated GFP was the highest (52%), which was significantly higher than that of SB (47%) and Tol2 (36%) (P0.05). However, in zebrafish embryos at 36 and 60 h (hour past fertilization,hpf after fertilization, the positive rates of GFP in Tol2 were 36% (Nf677) and 37% (Nf685), respectively. It was significantly higher than SB (28%N=691 and 27%N=708) and PB (30%N=687 and 32%N=675) (P0.05). Transposon mediated cytoplasmic microinjection could obtain higher transgene efficiency, and the transposon activity was different in species, and the gene transfer efficiency of PB transposon was the highest in mammalian embryos. Tol2 transposon has the highest gene transfer efficiency in fish embryos. This study provides an important reference for improving the efficiency of transgenic animals.
【作者单位】：扬州大学表观遗传学与表观基因组学研究所;扬州大学动物科学与技术学院;
【基金】：国家转基因重大专项重点课题子课题(No.2014ZX08006005-008) 江苏省产学研联合创新资金-前瞻性联合研究项目(No.BY2013063-03) 中国博士后科学基金(No.20100480371) 江苏省普通高校学术学位研究生科研创新计划项目(No.KYLX_1357)
【分类号】：Q78

【参考文献】