慢病毒介导的绿色荧光蛋白转基因小鼠的制备及鉴定

摘要

Objective To establish a technology platform of transgenic animals mediated by lentiviral vector through a practice of preparing the transgenic mice by lentiviral vector carrying green fluorescent protein (GFP). Methods 293FT cells were transfected by a mixture of leniviral vector FUGW and viraPower· Lentiviral packaging mix (1 ' 2) using Lipofectamine· 2000. After undergoing concentration, the titer of packaged lentivirus was tested in 293T cells. The transgenic mice were reproduced by injecting the lentiviral vectors into the perivitelline space. Integration of exogenous gene in transgenic mice was confirmed by PCR detection, and the expression of exogenous gene in transgenic mice was identified by fluorescence stereomicroscope. Results Twenty-four hours later, GFP expression was detected in 293FT cells. A great amount of green fluorescence can be detected under fluorescence inverted microscope after 72 hours. The transfection efficiency of 293FT cells reached to higher than 95%. The virus titer of packaged lentiviral vector was 106U/ml, and was 108U/ml after concentration. In Fo generation, GFP could be detected in 70. 3% (26/37) transgenic mice by PCR. Under fluorescence stereomicroscope, GFP expressed in 65.2% (15/23) and 55.0% (11/20) of F0 and F1 generation, respectively, and the level was comparable. The GFP expression was also widely observed in both Fo and Fi generations. Conclusions The transgenic mice model carrying GFP is produced and the GFP gene can be transmitted through germline cells. The technology platform of transgenic mice mediated by lentiviral vector has been successfully established.%目的 以携带绿色荧光蛋白(GFP)的慢病毒为载体制备转基因小鼠,建立慢病毒介导的转基因动物制备技术平台.方法 将慢病毒表达载体FUGW与ViraPowerTM Lentiviral Packaging Mix按1∶2比例混合,用LipofectamineTM 2000转染293FT细胞,包装出的病毒经浓缩后以293T细胞测定病毒滴度.采用受精卵卵周隙显微注射的方式制备转基因小鼠.出生小鼠用PCR法检测GFP基因整合情况,并在荧光体视显微镜下鉴定外源基因的表达情况.结果 病毒包装过程中,转染后24h即可见GFP表达,72h时293FT细胞的转染效率达95%以上,镜下可见大量绿色荧光.包装出的慢病毒滴度为106 U/ml,经浓缩后可达108U/ml.PCR检测显示,F0代小鼠GFP FCR阳性率为70.27%(26/37).荧光体视显微镜下观察荧光小鼠所占比例,F0代为65.2%(15/23),F1代为55.0%(11/20),荧光强度在两代个体中相当.F0代、F1代中GFP均呈广泛表达.结论制备出携带GFP可传代的转基因小鼠,建立了慢病毒载体介导的转基因小鼠制备技术平台.