Objective： The aim of the study was to construct miRNA-451 expression vector pLMP-miRNA-451 which could help identify the functions of miRNA-451 in SGC-7901 cell. Methods： Total RNA was extracted from SGC-7901 cells...Objective： The aim of the study was to construct miRNA-451 expression vector pLMP-miRNA-451 which could help identify the functions of miRNA-451 in SGC-7901 cell. Methods： Total RNA was extracted from SGC-7901 cells to synthesized cDNA. The synthesized cDNA encoding pre-miRNA-451 was amplified by polymerase chain reaction （PCR）. The PCR product was separated by electrophoresis on 1% agarose gel and then recovered and purified. The purified cDNA fragments of miRNA-451 precursor sequence was then ligated with vector pLMP for 1 h by using DNA ligase to form pLMP- miRNA-451 plasmid. After that, the pLMP-miRNA-451 plasmid was transformed into E. coli DH5a strain expression system to clone and amplificate. The purified pLMP-miRNA-451 extracted from E. coli DH5a via transformation and clone screening was identificatied with restriction enzyme digestion and DNA sequencing. At last, pLMP-miRNA-451 was transfected into SGC-7901 cells with lip2000. Real-time PCR was used for detection of the miRNA-451, the transfection efficiency was ob- served under fluorescence microscopy and cell counting kit-8 assay was conduced to evaluate the effect of miRNA-451 on SGC-7901 cell proliferation. Results： Our results showed that pLMP-miRNA-451 expression vector was not only constructed successfully and effectively infected SGC-7901 cells, but also could repress the SGC-7901 cell proliferation. Conclusion： The constructed plasmid pLMP-miRNA-451 could used for further studies of miRNA-451 in SGC-7901 cell lines.展开更多
MicroRNAs (miRNAs) are small endogenous RNAs molecules,approximately 21–23 nucleotides in length,which regulate gene expression by base-pairing with 3′ untranslated regions (UTRs) of target mRNAs.However,the functio...MicroRNAs (miRNAs) are small endogenous RNAs molecules,approximately 21–23 nucleotides in length,which regulate gene expression by base-pairing with 3′ untranslated regions (UTRs) of target mRNAs.However,the functions of only a few miRNAs in organisms are known.Recently,the expression vector of artificial miRNA has become a promising tool for gene function studies.Here,a method for easy and rapid construction of eukaryotic miRNA expression vector was described.The cytoplasmic actin 3 (A3) promoter and flanked sequences of miRNA-9a (miR-9a) precursor were amplified from genomic DNA of the silkworm (Bombyx mori) and was inserted into pCDNA3.0 vector to construct a recombinant plasmid.The enhanced green fluorescent protein (EGFP) gene was used as reporter gene.The Bombyx mori N (BmN) cells were transfected with recombinant miR-9a expression plasmid and were harvested 48 h post transfection.Total RNAs of BmN cells transfected with recombinant vectors were extracted and the expression of miR-9a was evaluated by reverse transcriptase polymerase chain reaction (RT-PCR) and Northern blot.Tests showed that the recombinant miR-9a vector was successfully constructed and the expression of miR-9a with EGFP was detected.展开更多
基金supported by a grant from the Natural Science Foundation of China (No. 8100098)
文摘Objective： The aim of the study was to construct miRNA-451 expression vector pLMP-miRNA-451 which could help identify the functions of miRNA-451 in SGC-7901 cell. Methods： Total RNA was extracted from SGC-7901 cells to synthesized cDNA. The synthesized cDNA encoding pre-miRNA-451 was amplified by polymerase chain reaction （PCR）. The PCR product was separated by electrophoresis on 1% agarose gel and then recovered and purified. The purified cDNA fragments of miRNA-451 precursor sequence was then ligated with vector pLMP for 1 h by using DNA ligase to form pLMP- miRNA-451 plasmid. After that, the pLMP-miRNA-451 plasmid was transformed into E. coli DH5a strain expression system to clone and amplificate. The purified pLMP-miRNA-451 extracted from E. coli DH5a via transformation and clone screening was identificatied with restriction enzyme digestion and DNA sequencing. At last, pLMP-miRNA-451 was transfected into SGC-7901 cells with lip2000. Real-time PCR was used for detection of the miRNA-451, the transfection efficiency was ob- served under fluorescence microscopy and cell counting kit-8 assay was conduced to evaluate the effect of miRNA-451 on SGC-7901 cell proliferation. Results： Our results showed that pLMP-miRNA-451 expression vector was not only constructed successfully and effectively infected SGC-7901 cells, but also could repress the SGC-7901 cell proliferation. Conclusion： The constructed plasmid pLMP-miRNA-451 could used for further studies of miRNA-451 in SGC-7901 cell lines.
基金Project supported by the National Basic Research Program (973) of China (No. 2005CB121004)the National High-Tech R & D Program (863) of China (No. 2006AA10A119)+1 种基金the Innovation Foundation for Graduate Students of Jiangsu Provincethe National Natural Science Foundation of China (No. 61001013)
文摘MicroRNAs (miRNAs) are small endogenous RNAs molecules,approximately 21–23 nucleotides in length,which regulate gene expression by base-pairing with 3′ untranslated regions (UTRs) of target mRNAs.However,the functions of only a few miRNAs in organisms are known.Recently,the expression vector of artificial miRNA has become a promising tool for gene function studies.Here,a method for easy and rapid construction of eukaryotic miRNA expression vector was described.The cytoplasmic actin 3 (A3) promoter and flanked sequences of miRNA-9a (miR-9a) precursor were amplified from genomic DNA of the silkworm (Bombyx mori) and was inserted into pCDNA3.0 vector to construct a recombinant plasmid.The enhanced green fluorescent protein (EGFP) gene was used as reporter gene.The Bombyx mori N (BmN) cells were transfected with recombinant miR-9a expression plasmid and were harvested 48 h post transfection.Total RNAs of BmN cells transfected with recombinant vectors were extracted and the expression of miR-9a was evaluated by reverse transcriptase polymerase chain reaction (RT-PCR) and Northern blot.Tests showed that the recombinant miR-9a vector was successfully constructed and the expression of miR-9a with EGFP was detected.
