应用双色荧光原位杂交技术对转基因小鼠进行整合位点的染色体定位  

Chromosomal localization of foreign genes in transgenic mice using dual-color fluorescence in situ hybridization

作　　者：林丹[1] 龚秀丽[1] 李伟[1] 郭歆冰[1] 朱怡文[1] 黄英[1] 

机构地区：[1]上海交通大学医学遗传研究所,卫生部医学胚胎分子生物学重点实验室,200040

出　　处：《中华医学遗传学杂志》2008年第1期40-44,共5页Chinese Journal of Medical Genetics

基　　金：国家973项目（2004CB518806）

摘　　要：目的建立一种高度灵敏、特异的双色荧光原位杂交（dual-color fluorescence in situ hybridization,D-FISH）技术，对两种双阳性转基因小鼠外源基因进行整合位点的染色体定位。方法对一只整合单纯疱疹病毒胸苷嘧啶激酶（herpes simplex virus thymidine kinase, HSV-tk）／增强型绿色荧光蛋白（ enlmneed green fluorescence protein, eGFP）的转基因小鼠以及两只整合RNA干扰载体（ RNA interference, RNAi） 的β^654地中海贫血模型小鼠进行实验，脾脏细胞经培养后获得中期分裂相标本片，各加入适量生物素、地高辛标记探针混合液杂交，分别用罗丹明红色荧光抗体及FITC绿色荧光抗体进行D-FISH检测。结果两种转基因小鼠均能在同一个分裂相上同时检测到双色荧光信号。其中，HSV-tk／eGFP双阳性小鼠的分裂相上出现较强的绿色HSV-tk信号和红色eGFP信号，分别定位于染色体2E5-G3及8A2-A4；β^654／RNAi双阳性小鼠检测到红色β^654荧光信号及绿色RNAi荧光信号。经定位分析，β^654均整合在染色体7D3-E2，RNAi病毒载体则是随机整合，其中一只鼠主要整合在12B1位点，而另一只鼠主要整合在染色体1E2．3-1F、3A3两个位点。结论用自行制备的DNA探针建立了高度灵敏、特异的D-FISH技术，同时结合G显带对双阳性转基因小鼠进行染色体基因定位。该技术平台的建立对于转基因动物和基因治疗动物模型的研究具有非常重要的意义。Objective To establish a highly sensitive and specific dual-color fluorescence in situ hybridization （D-FISH） method used for chromosomal localization of foreign genes in double transgenic mice. Methods Two strains of double transgenic mice were used in this experiment, one was integrated with the herpes simplex virus thymidine kinase （HSV-tk） and the enhanced green fluorescence protein （eGFP）, the other was with the short hairpin RNA interference （RNAi）and β^654. Splenic cells cultured in vitro were arrested in metaphase by colchicine and hybridized with digoxigenin-labeled and biotinylated DNA probes, then detected by rhodamine-conjugated avidin and FITC-conjugated antidigoxigenin. Results Dual-color fluorescence signals were detected on the same metaphase in both transgenic mice strains. In HSV-tk/eGFP double transgenic mice, strong green fluorescence for HSV-tk and red for eGFP were observed and localized at 2E5-G3 and 8A2-A4 respectively. In β^654/RNAi mice,β^654 was detected as red fluorescence on chromosome 7D3-E2, and RNAi showed random integration on chromosomes. It was detected as green fluorescence on chromosome 12B1 in one mouse, while on 1E2.3-1F and 3A3 in the other. Conclusion Highly sensitive and specific D-FISH method was established using the self-prepared DNA probes, and chromosomal localization of the foreign genes was also performed in combination with G-banding in double wansgenie mice. This technology will facilitate the researches in transgenic animals and gene therapy models.

关 键 词：转基因小鼠 整合位点 双色荧光原位杂交 染色体定位 

分 类 号：Q78[生物学—分子生物学]