应用抑制性消减杂交技术筛选脂肪细胞分化相关基因

Screening of differentially expressed genes during adipocyte differentiation by suppression subtractive hybridization technique

目的:利用脂肪分化细胞模型筛选与脂肪细胞分化相关的差异性表达基因。方法:采用抑制性消减杂交技术,以诱导分化后的人前体脂肪细胞系SW872总RNA为Tester,诱导分化前的人前体脂肪细胞系SW872为Driver,将消减杂交获得的DNA片段与pGM-T载体连接,构建cDNA文库后,转化大肠杆菌DH5α,筛选阳性克隆。将获得的阳性克隆插入片段进行测序分析,并与GenBank数据库进行同源性分析,获得差异性表达基因。结果:扩增消减cDNA文库获得135个白色克隆,随机挑选64个进行测序,共获得34个阳性克隆基因。结论:本研究成功构建了脂肪细胞分化差异性表达基因的消减cDNA文库,筛选出了与脂肪细胞分化相关的差异表达基因。

Objective： To screening differentially expressed genes related to adipoeyte differentiation. Methods： Total RNA extracted from the preadipocyte cell line SW872 was taken as the Driver and the total RNA from the differentiated adipocytes SW872 as the Tester. Suppression subtractive hybridization （SSH） was used to isolate the eDNA fragments of differentially expressed genes. The products of SSH were inserted into pGM-T vector to establish the subtractive library. The library was amplified through E. coli transformation and positive clones of the transformants were screened. Positive clones were sequenced. Nucleic acid similarity was subsequently analyzed by comparing with the data from GenBank. Results： There were 135 white clones in the cDNA library ,64 positive clones were chosen randomly and sequenced and similarity search revealed 34 genes which expressed differentially in adipocyte differentiation. Conclusion： The subtracted cDNA library for differentially expressed in adipocyte differentiation has been successfully constructed and the interesting candidate genes related to adipocyte differentiation have been identified.