Regulatory effect of human beta-interferon matrix attachment region on transgene expression in CHO cells
- VernacularTitle:人?-干扰素核基质附着区在CHO细胞中对转基因表达的调控
- Author:
Yuxi ZAN
;
Tianyun WANG
;
Junhe ZHANG
;
Li WANG
- Publication Type:Journal Article
- From:
Chinese Journal of Tissue Engineering Research
2007;0(29):-
- CountryChina
- Language:Chinese
-
Abstract:
BACKGROUND: Matrix attachment region (MAR), a DNA sequence, is still bound to the nuclear matrices after chromatindigested with restriction endonuclease, not only affects expression of endogenous gene, but also overcames transgenic silence andimproves transcription and expression of exogenous gene. OBJECTIVE: To investigate the influence of ?-interferon MAR of CHO cells on the transgenic expression of chloramphenicolacetyltransferase (CAT). DESIGN, TIME AND SETTING: The opening experiment was performed at the Department of Biochemistry and MolecularBiology, Molecular Institute, Xinxiang Medical College from October 2006 to April 2007. MATERIALS: CHO cell lines were obtained from China Center for Type Culture Collection. The pCATG vector of CAT and G418screening markers were constructed by this laboratory. METHODS: Human ?-interferon MAR by PCR was digested with SacI/KpnI and BamHI/SalI, and was inserted into pCATGvector, which was propagated in Escherichia coli JM109, then extracted and purified followed by enzyme digestion analysis. Vectorof CAT expression cassette and human ?-interferon MAR by the two sides was successfully constructed, and christened aspCAT-MAR. Two methods were compared between CHO cells of pCATG transformation and CHO cells of pCATG-MARtransformation. After G418 selecting, genome DNA of cell lines of G418 was extracted, then primers for PCR to amplify the CATtarget gene fragment was designed. MAIN OUTCOME MEASURES: The activity of CAT was analyzed by ELISA method. It was also tested to see if thepCATG-MAR was stably integrated into genomic DNA in the transfected cells. RESULTS: CHO cells of pCATG transformation was screened to have 16 strains of positive cell, and CHO cells of pCATG-MARtransformation was screened to have 17 strains of positive cell. Human ?-interferon MAR could increase the CAT gene expressionby 2.8 fold. The coefficient of variation of CHO cells of pCATG transformation was 2.065 0, and coefficient of variation of CHOcells of pCATG-MAR transformation was 0.813 1. Genome DNA of stable transformation cell lines was amplified by a fragment of437 bp. The results confirmed the pCAT-MAR vector was stably integrated into genomic DNA. CONCLUSION: Human ?-interferon MAR can increase transgenic expression in CHO cells and decrease the transgenicexpression variation in different transfected cells.
