慢病毒载体法制备红色荧光蛋白转基因小鼠  

mRFP Transgenic Mice Generated by Lentivirus-Mediated Gene Delivery

作　　者：贾俊双[1] 肖高芳[1] 林晓琳[1] 张晟[2] 姚志芳[1] 杜文婷[1] 申红芬[1] 刘科[3] 饶子亮[3] 孙妍[1] 姚开泰[1] 肖东[1,2] 

机构地区：[1]南方医科大学肿瘤研究所,广州510515 [2]南方医科大学比较医学研究所暨实验动物中心,广州510515 [3]广东省医学实验动物中心,广州528248

出　　处：《中国比较医学杂志》2010年第2期12-16,I0001,共6页Chinese Journal of Comparative Medicine

基　　金：国家自然科学基金委员会-广东省联合基金重点项目(u0732006);国家自然科学基金项目(30271177);广东省自然科学基金项目(9151063101000015);广东省科技计划项目(2009B060300008);广东省医学科学技术研究基金(A2007359);南方医科大学优秀中青年科技人才库科研资助金;广州地区科学仪器协作共用网专用基金(2006176)

摘　　要：目的利用慢病毒介导的转基因方法制备红色荧光蛋白(mRFP)转基因小鼠,并建立转基因小鼠的技术平台。方法将携带mRFP基因的慢病毒注入ICR鼠单细胞受精卵卵周隙以感染受精卵,胚胎移植进假孕母鼠以获得仔代鼠,然后应用小动物活体成像仪、体视荧光显微镜和PCR等鉴定并获得mRFP转基因鼠。结果移植卵周隙注射有慢病毒的胚胎40枚给2只假孕母鼠,共获得仔鼠6只;利用体视荧光显微镜检测mRFP表达,在蛋白水平证实6只F0代中,2只(R3和R4)鼠耳高表达mRFP,其余的弱表达mRFP(R1、R2和R5)或荧光强度(R6)与野生型ICR鼠无明显差别,而DNA水平检测证实,6只F0代中,5只(R1、R2、R3、R4和R5)基因组中整合有外源转基因hUb-mRFP,预示基因型鉴定结果很好验证了体视荧光显微镜鉴定结果。此外,mRFP转基因首建鼠基因组中整合的mRFP基因可稳定遗传和表达。结论建立了慢病毒法快速制备转基因小鼠的技术平台,这为针对不同基因建立相应转基因小鼠以实现恒定或条件性的转基因过表达或RNA干涉(RNAi),并进而在体内解析相应基因功能和建立人类疾病模型等奠定了坚实基础。Objective To establish a technologic platform for generating transgenic mice by lentivirus-mediated delivery of foreign gene（s） into zygotes.Methods／Monomeric red fluorescent protein（mRFP） transgenic mice were generated by subzonal injection of lentivirus harboring mRFP gene into single-cell fertilized eggs of ICR mice to infect fertilized eggs,and subsequently the embryos infected by lentivirus were transplanted into the pseudopregnant mice to attain F0 mice,followed by screening mRFP transgenic mice from potential founders via mRFP assay by using small animal in vivo imaging system or under stereo fluorescence microscope at birth 3 weeks after birth,and the results of mRFP assay were subsequently confirmed by PCR for genotyping.Results 40 virus-injected eggs were transplanted into 2 pseudopregnant mice and attained 6 potential transgenic founders（i.e.,R1,R2,R3,R4,R5 and R6）.When observed under fluorescent stereo microscope,the ears from R3 and R4 showed strong red fluorescence,while the ears from R1,R2 and R5 showed relatively weaker signals and the ear from R6 showed almost no signal.PCR-based genotyping demonstrated that mRFP gene was actually integrated into the genome of F0 mice（i.e.,R1,R2,R3,R4 and R5）,but not R6,as confirmed the results of mRFP assay.Moreover,mRFP transgene could be transmitted from founders（R3 and R4） to subsequent generation（F1 progeny）.Conclusions／The mRFP transgenic mice have been efficiently and rapidly generated by subzonal injection of lentiviruses into single-cell fertilized eggs of ICR mice.It has laid a foundation for further experimental researches on many human diseases.

关 键 词：红色荧光蛋白 慢病毒载体法 转基因小鼠 

分 类 号：Q78[生物学—分子生物学]