小鼠FSP27基因沉默的前脂肪细胞系的建立

用含有针对小鼠FSP27基因的siRNA慢病毒,感染小鼠前脂肪细胞系3T3-L1,建立小鼠FSP27基因沉默的前脂肪细胞系,为进一步研究该基因在脂肪细胞分化和脂肪代谢过程中的作用提供实验材料.根据小鼠FSP27基因设计双链siRNA,克隆至pSilencer2.1-U6质粒,形成含U6-siRNAbox的重组质粒.在293T细胞中检测siRNA沉默FSP27基因的效率,结果显示,siRNA可以高效地抑制外源小鼠FSP27的表达.将U6-siRNAbox重组到慢病毒载体FG12上,并将慢病毒载体与其他辅助载体用磷酸钙法转入293T细胞,包装成慢病毒.收集、浓缩、纯化病毒上清并用来感染靶细胞3T3-L1,检测感染效率和内源蛋白表达量的变化.结果显示,该siRNA可以高效地抑制内源小鼠FSP27的表达,并且siRNA插入基因组中,形成稳定表达.至此,小鼠FSP27基因沉默的前脂肪细胞系成功建立,为研究FSP27基因的功能提供了研究基础.

Establishment of FSP27 Gene Knockdown Preadipocyte Cell Line

Obesity and its related metabolic diseases become major health problems in the world.Adipose tissue plays an important role in the development of obesity.FSP27,a member of the CIDE family proteins,is expressed at high levels in white adipose tissue and differentiated 3T3L1 cells.The objective of current study is to establish a FSP27 knockdown preadipocyte cell line to investigate the in vivo function of mouse FSP27.The double strand siRNA of mouse FSP27 corresponding to nucleotides 270 to 291 was synthesized and inserted into pSilencer2.1.pSilencer-siFSP27 was co-transfected into 293T cells with the HA-mFSP27 expression vector to test its knock-down efficiency.The FSP27 siRNA was then transferred to a lentiviral vector.Lentivirus were generated and used to infect 3T3-L1 cells.It was shown here that lentivirus containing FSP27siRNA can effectively knockdown FSP27 expression in 3T3-L1 cells.Establishment of FSP27 knock-down cell line provides a useful tool for the study of in vivo function FSP27.