双筛选标记打靶载体的构建及其功能鉴定

Construction and functional analysis of a common gene targeting vector with double-selection markers

摘要:

利用DNA同源重组方法 (基因打靶) 对动物基因组进行修饰是转基因研究的重要手段之一。为了构建一个高效的通用型基因打靶载体，本研究以pBS246质粒为骨架，在两个LoxP序列之间插入正筛选标记新霉素磷酸转移酶(neo)基因和绿色荧光蛋白 (EGFP) 基因；在两个LoxP序列外侧分别插入两组携带“8碱基”酶切位点的多克隆位点序列 (MCS-1和MCS-2) 和负筛选标记单纯疱疹病毒胸苷激酶 (HSV-tk) 基因，构建成通用型基因打靶载体pGT-V1，并且在C2C12细胞中验证了载体中各个元件的功能。该载体具有如下特点：1) 在载体中引入绿色荧光标记，可以实时监控载体的转染效率，而转染效率的提高为高效基因打靶提供了保证；2) 在两个LoxP位点间插入绿色荧光标记，可以直观监测打靶后遗留的筛选标记的去除情况，并且可以通过流式细胞仪或免疫磁珠法，将最终去除了筛选标记的阳性细胞 (即丢失绿色荧光的细胞) 分选出来，降低筛选标记在中靶细胞中可能产生的负面影响；3) 采用“8碱基”酶切位点的MCS序列，便于DNA大片段的连接和重组，极大提高了该载体的通用性。总之，该载体优化了基因打靶的技术手段，为有效开展基因打靶和转基因动物研究提供了新平台。

Abstract:

Homologous recombination is an important technique that is used to modify mammalian genome. Here, we constructed an efficient common gene targeting vector based on the plasmid pBS246. The vector consisted two positive selection markers, neomycin resistance gene (neo) and enhanced green fluorescent protein gene (EGFP) flanked by locus of X-over P1 (LoxP) sites. Two synthesized multiple cloning sequences MCS-1 and MCS-2 that contain several “8 bp cutter” enzyme sites were placed in outside of LoxP sites. Additionally, a negative selection marker HSV-tk (herpes simplex virus thymidine kinase) gene was located adjacent to MCS-1 site. The constructed vector was named pGT-V1, and its functions were characterized in C2C12 cells. The vector had the following unique features: 1) EGFP was used to monitor instantly the transfection rate that was essential for increasing the efficiency of gene knockout (KO); 2) The EGFP marker located between two LoxP sites was able to be removed from KO positive cells to avoid the potential damage of selection markers to the recipient cells. The process could be monitored visually and the positive cells without selecting markers (the loss of green fluorescent cells) could be sorted out by either flow cytometry or immunomagnetic beads; 3) “8 bp cutter” restriction sites were embedded in MCS sequences, which then enhanced the versatility of this vector. In summary, the constructed plasmid optimized the vector of gene targeting and provided a new technique means for the transgenic animal research.