PDGF-C 3'UTR区荧光素酶报告基因载体构建

3'UTR of PDGF-C Dual-luciferase Reporter Gene Vector

目的:构建不同长度包含目的片段的PDGFC 3`UTR双荧光素酶报告基因载体,为进一步研究PDGF-C mRNA的上游miRNA调节做准备。方法:培养人乳腺癌细胞系MDA-MB-231细胞,提取腺癌细胞系MDA-MB-231细胞的基因组DNA,以提取的基因组为模板,通过PCR扩增不同长度的PDGFC 3`UTR片段,经胶回收后,将回收的目的片段插入报告基因载体SV40-p GL3中,再经转化将克隆好的载体转入细菌内扩增(先在固体培养基上扩增为菌落,然后再接种进液体细菌培养管中扩增),扩增细菌后进行质粒提取,并进行菌落PCR及双酶切鉴定,最后送公司进行基因序列检测鉴定。结果:成功构建了不同长度目的片段的PDGFC 3`UTR的双荧光素酶报告基因载体。结论:本实验构建了不同长度的PDGF-C mRNA的3’UTR区的双荧光素酶报告基因载体,为进一步研究PDGF-C mRNA的上游miRNA调节打下了基础。

Objective： To construct dual lucifemse reporter gene vector containing different length of PDGFC-3＇-UTR sequence and lay foundation for the regulation study of the upstream miRNA of PDGF-C. Methods： First, Genomic DNA was extracted from cultured breast cancer cell line MDA-MB-231. Then different lengths of PDGFC-3＇-UTR sequence were amplified by PCR, Inserted into the reporter gene vector SV40-pGL3, and transformed into competent E.coli cells for further propagation under the selection of appropriate antibiotics. Finally, the recombinant plasmid extracted from bacterial pellet was subjected to double enzymatic restriction analysis and Sanger sequencing. Results： In this experiment, dual luciferase report gene vectors for different PDGFC-3 ＇-UTR sequences were constructed successfully. Conclusions： The constructed dual-luciferase Reporter gene vetors for 3＇-UTR of PDGFC gene might lay foundation for further study of upstream miRNA ofPDGF-C mRNA.