Abstract:

BACKGROUND: Matrix attachment region (MAR) are DNA elements that bound to the nuclear matrices after chromatin digested with restriction endonuclease. Plenty of studies have shown that MAR considered as initial point of DNA replication or transcription of regulatory gene. Thereby, construction of MAR expression vector can elevate the overall level of transgene expression, enhance stability of exogenous gene, as well as increase frequency of stable transfectant cells.
OBJECTIVE: To construction pLXSN-CAT recombinant retrovirus vector that containing chloramphenicol acetyltransferase (CAT) via cloning MAR sequence of human, and to explore the influence of MAR on the gene expression.
METHODS: An open experiment was performed at the Department of Biochemistry and Molecular Biology, Xinxiang Medical College from September 2007 to December 2007. The PLXSN-CAT vector of CAT was constructed by the laboratory. TaqDNA polymerase, T4 DNA ligase, DNA Marker, restriction enzyme BamHⅠ, agarose gel DNA purification kit, as well as plasmid purification kit were purchased fromTakara Biotechnology (Dalian) Co., Ltd. The sequence of csp-B MAR was amplified by polymerase chain reaction (PCR) method applied to human DNA. The fragment was inserted into retrovirus vector PLXSN-CAT plasmid. The recombinant plasmid was verified by double digestion and DNA sequencing.
RESULTS AND CONCLUSION: The length of specific fragment applied by PCR was 931 bp, and the recombinant plasmid PLXSN-CAT-MAR presented two bands: 5.9 kb and 931 bp using respective restriction enzymes BamHⅠ. The sequence of MAR was confirmed by blasting to Genbank (serial numobr: M62716). It suggested that MAR had been cloned into PLXSN-CATR vector correctly. The recombinant retrovirus vector PLXSN-MAR was successfully constructed.