Construction of C_(57)BL mice survivin gene miRNA expression vector
10.3969/j.issn.1673-8225.2009.53.019
- VernacularTitle:C_(57)BL小鼠survivin基因miRNA表达载体的构建
- Author:
Haifeng WANG
;
Xin GOU
;
Yanmin LI
;
Weiyang HE
;
Huaan YANG
- Publication Type:Journal Article
- From:
Chinese Journal of Tissue Engineering Research
2009;13(53):10491-10494
- CountryChina
- Language:Chinese
-
Abstract:
BACKGROUND: Increased expression of survivin in various tumor tissues can regulate cell proliferation, division, and plays an important role in protecting cells from apoptosis.OBJECTIVE: To construct the specific micro RNA (miRNA) expression vector that can block the C_(57)BL mice survivin gene by RNA interference (RNAi) technique.DESIGN, TIME AND SETTING: The single sample observation was performed at the experimental center of Department of Neurology, The First Afliliated Hospital of Chongqing Medical University from June to November 2008.MATERIALS: Ring-shaped pcDNA~(TM)6.2-GW/EmGFPmiR and BLOCK-iT~(TM) Pol II miR RNA interfered Expression Vector Kit with EmGFP was produced by Invitrogen Company. DH5a E. coli was preserved at the laboratory. Xho I and BamH I enzyme, spectinomycin were provided by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd.METHODS: According to sequence of mRNA of C_(57)BL mice survivin provided by Genebank, four pairs of specific oligonucleotide sequences were designed and synthesized by using the software. The annealed oligonucleotide fragment was sub-cloned into pcDNA~(TM)6.2-GW/EmGFPmiR expression vector by gene clone technique and transformed into DH5a E. coli, subsequently, a single colony was incubated into liquid medium containing spectionmycin. Finally the plasmid was extracted.MAIN OUTCOME MEASURES: The recombinant vector was identified by sequencing and agarose gel electrophoresis.RESULTS: The sequencing revealed that insertion element was correctly cloned into the vector without nucleotide mutation, absence or insertion abnormality. The result of double enzyme digestion demonstrated that the fragment length was coincidence with expectation.CONCLUSION: The C_(57)BL mice survivin miRNA expression vector is successfully constructed.
