摘要
目的研究外源基因在转基因小鼠及其家系中的整合状况。方法采用PCR、定量PCR和荧光原位杂交(FISH)的方法对原代转基因小鼠中外源人凝血因子Ⅸ(hFⅨ)基因的整合和嵌合情况进行分析。使用PCR、直接测序法鉴定整合的外源基因多拷贝的连接方向和连接方式。结果7个家系中F0-8、F0-10和F0-11的后代中阳性个体比例明显低于50%,3个原代小鼠DNA中hFⅨ基因拷贝数分别只有子代中的66.2%、18.8%和28.3%。F0-11小鼠各脏器中嵌合比差异极大,且未见胚系特异性分布。不同个体间整合拷贝数差异极大,最少的F0-69为单拷贝,最多的F0-10有43个拷贝。而整合位点分析发现外源基因在随机分布中仍呈现一定的趋向性。PCR和测序结果证明所有小鼠中外源基因多拷贝均为头尾连接,连接的机制以粘性末端介导的连接为主,F0-13中还存在同源序列配对、断裂、修复介导的头尾连接。结论在整合有hFⅨ基因的转基因小鼠中多拷贝外源基因多以粘性末端介导的头尾连接方式整合在染色体的某些特定区域。
Objective To invesigate the integration status of the foreign gene in transgenic mice lines. Methods The integration and mosaic status of foreign human coagulation factor IX (hFIX) gene in the transgenic mice was analyzed with PCR, real-time PCR and fluorescence in situ hybridization (FISH) . The patterns of the juncture and direction in multi-copy integrated foreign gene was detected by PCR and sequencing. Results In seven hFIX transgenic mice lines, the transgene positive rates in the offspring of the founders ( F0-8, F0-10 and F0-11 ) were theoretically much lower than 50%. The copy numbers of foreign hFIX gene in these founders were only 66.2% , 18.8% and 28.3% of that in the offspring, respectively. Real-time quantitative PCR analysis in F0-11 showed variant integrated gene copies in different organs, and revealed no specific distribution in germ tissues. The copy number of the integrated foreign gene in different transgenic mice was also quite different. The highest was 43 copies in F0-10, while the lowest had only onecopy in F0-69. The integration sites of the foreign gene on chromosomes were random but some hot spots could be observed. PCR and sequencing analysis indicated that hFIX gene integrated in 9 transgenic mouse lines were arrayed head-to-tail, mainly by viscous ends. Other mechanisms for multi-copy integration of foreign gene such as homologous pairing, fragment breakage and repair were also identified in F0-13. Conclusion The molecular patterns of multi-copy integration in transgenic mice were often arrayed head-to-tail and linked with viscous ends in some specific regions of chromosomes.
出处
《医学分子生物学杂志》
CAS
CSCD
2006年第6期407-413,共7页
Journal of Medical Molecular Biology
基金
上海市现代生物与新药产业发展基金(No.024319206)
关键词
转基因小鼠
外源基因
整合
拷贝数
嵌合体
transgenic mouse
foreign gene
integration
copy number
mosaic