Cloning of human REG ? cDNA and construction of the stable transfective cell line / 重庆医科大学学报

ABSTRACT

Objective:To construct the stable transfective cell line with the eukaryotic expression vector for human REG?cDNA to laid the foundation for further study of the function of REG?.Methods:REG?cDNA was obtained by RT-PCR of total RNA extracted from the human breast cancer cell MCF-7,and it was digested by the restriction endonuclease EcoRⅠand EcoRⅤbefore connection withPcDNA3.1.The recombination with forward insert were slelected by restriction endonuclease digestion and confirmed correct by sequencing and then transfected into HBL-100 cell by using lipofectamine2000.The stable transfected cell line was selected in medium containing antibiotic(G418).Results:The result of immunocytochemistry and RT-PCR of total RNAextracted from the stable transfected cell line showed that there existed the overexpression of REG?cDNA in it.Conclusion:The construction of the eukaryotic expression vector for REG?was successful,and the stable transfected cell line for overexpression REG? was obtained by G418 selection.This work may pave the way for further study of the functions of REG?.