胎儿间充质干细胞cDNA扣除文库的构建

Construction of Fetal Mesenchymal Stem Cell cDNA Subtractive Library

为了获得胎儿和成人间充质干细胞 (mesenchymalstemcells,MSC)的差异表达基因 ,尤其是在胎儿MSC中特异表达的基因 ,应用抑制扣除杂交技术构建了胎儿和成人MSCcDNA扣除文库。首先 ,分别从胎儿和成人MSC中提取总RNA ,按照SMARTPCR方法依次合成单链和双链cDNA ,经RsaⅠ酶切后 ,胎儿MSCcDNA分别与两种不同的接头连接 ,再与成人MSCcDNA经过两次扣除杂交及两轮抑制性PCR扩增后 ,将产物与T载体连接构建成cDNA扣除文库 ,并转染大肠杆菌进行文库扩增。结果 :文库扩增后共获得 890个克隆 ,对所获得的克隆进行PCR扩增鉴定 ,获得 76 8个阳性克隆 ,阳性率为 86 .3%。插入片段大小分布在 0 .2 - 1kb ,平均为 4 0 0 - 6 0 0bp。结论 :抑制扣除杂交技术是一种简便、有效的筛选差异表达基因的方法。胎儿MSCcDNA扣除文库的构建为进一步筛选、克隆胎儿MSC中特异性表达的功能相关基因奠定了基础。

To identify differentially expressed genes between fetal mesenchymal stem cell (MSC) and adult MSC, especially specified genes expressed in fetal MSC, a cDNA subtractive library of fetal MSC was constructed using suppression subtractive hybridization (SSH) technique. At first, total RNA was isolated from fetal and adult MSC. Using SMART PCR synthesis method, single strand and double strand cDNAs were synthesized. After RsaⅠ digestion, fetal MSC cDNAs were divided into two groups and ligated to adaptor 1 and adaptor 2 respectively. Results showed that the amplified library contains 890 clones. Analysis of 890 clones with PCR demonstrated that 768 clones were positive. The positive rate is 86.3%. The size of inserted fragments in these positive clones was between 0.2-1 kb, with an average of 400-600 bp. Conclusion: SSH is a convenient and effective method for screening differentially expressed genes. The constructed cDNA subtractive library of fetal MSC cDNA lays solid foundation for screening and cloning new and specific function related genes of fetal MSC.