ABSTRACT
Objective To construct recombinant lentivirus with the gene STAT3 of the Mus musculus,measure the expres-sion of STAT3,and conduct lentivirus packing and identification.Methods The mRNA of mouse myoblast was extracted and transformed into STAT3 cDNA by the special primer.then,STAT3 cDNA was amplified and reclaimed and inseted into pLVX-IRES-ZsGreen1 vector.Cleavage map and sequencing analysis were used for identification of the recombinant lentivirus vector (pLVX-IRES-ZsGreen1-STAT3).293 T cells were transfected with main vector pLVX-IRES-ZsGreen1-STAT3.and 48 h later, Western blott detected the expression of STAT3 protein.Lentiviral vectors were packaged and the titer was determined.Results The lentiviral vector plasmid pLVX-IRES-ZsGreen1-STAT3 was identified correctly by cleavage map and Co-transfection of 293 T cells with 48 h,the expression of STAT3 was significantly enhanced by western blot.And DNA sequencing analysis confirmed that STAT3 gene sequencing was exactly the same with that reported by genbank.Conclusion Lentiviral vector carrying STAT3 was successfnlly constructed and could express STAT3 with high efficiency,and can be used in further study.
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.
ABSTRACT
Objective:To construct eukaryotic expression vectors with the pSilencer2.0 vector for inhibiting human survivin gene by RNA interference,and to detect the effect of the silenced survivin gene on EJ cells.Methods:Two target gene segments were synthesized and cloned into the pSilencer2.0 vector respectively to construct two recombinant eukaryotic expression vectors,pSilencer2.0-SVV1and pSilencer2.0-SVV2,which were identified by enzyme digestion analysis and DNA sequencing.Then the EJ cells were transfected with the recombinant vectors by lipofection and the interference effect was detected by RT-PCR and Western-blot.Results:Enzyme digestion analysis and DNA sequencing showed that two target segments were cloned into pSilencer2.0 vectors correctly.The results of RT-PCR and Western blot indicated that pSilencer2.0-SVV1 and pSilencer2.0-SVV2 vectors could significantly knock down the transcription and expression of survivin gene(P