摘要
目的构建带有快速筛选基因标记的逆转录病毒载体。方法构建携带绿色荧光蛋白（ＧＦＰ）基因的逆转录病毒载体（ＧＣＧＦＰＰＸＳＮ），转染包装细胞系ＰＡ３１７，荧光显微镜及流式细胞仪观察结果。结果构建了一个携带快速筛选标记ＧＦＰ的逆转录病毒载体，其转染包装细胞系后在荧光显微镜及流式细胞仪（ＦＡＣＳ）中直接观察到该基因表达。应用该包装细胞系转染Ｔ细胞后第２天即表达绿色荧光蛋白，并可用ＦＡＣＳ进行分选。结论携带ＧＦＰ的逆转录病毒载体能够有效转染哺乳类动物细胞，与新霉素筛选基因相比，具有快速。
Objective To construct retroviral vector carrying rapidly selective marker. Methods The recombination retroviral vector GCGFPPXSN was constructed by cloning the green fluorescent protein(GFP) cDNA into the retroviral vector containing putative internal ribosome entry sites GCXPXSN and transferred in ecotropic packaging cell line PE501 by electroporation method.The supernatants of the PE501GCGFPPXSN were used to infect the amphotropic packaging cell line PA317.The G418 resistant clones were selected in 4 weeks and were detectable by fluorescence microscopy or by fluorescenceactivated cell sorting (FACS). Results A recombination retroviral vector GCGFPPXSN carrying rapidly selective marker GFP was constructed.GFP expression in packaging cell line PA317GCGFPPXSN transferred by GCGFPPXSN was detected by fluorescence microscopy of FACS.PA317GCGFPPXSN grew to a titer of 1.2×105 cpu/ml.By cocultivating retroviral vector producing cells and T lymphocytes,expression of GFP was observed in T lymphocytes 2 days after the end of the cocultivation.T lymphocytes expressing GFP were separated. Conclusion The mammalian cell can be efficient gene transfected by retroviral vector carrying GFP.The use of GFP for cell marking represents an important advantage over conventional strategies which typically involve the use of neomycin resistance.GFP, in fact,allows a rapid in vitro selection of transduced cell by FACS.The selection requires only twoday culture with this retroviral vector,compared with 10-14 day culture with a classical retroviral vector.
出处
《中华医学遗传学杂志》
EI
CAS
CSCD
北大核心
1998年第4期232-234,共3页
Chinese Journal of Medical Genetics
基金
国家自然科学基金
国家自然科学基金
国家及上海市博士后基金
