Regulatory effect of human beta-interferon matrix attachment region on transgene expression in CHO cells

Zan Yu-xi, Wang Tian-yun, Zhang Jun-he, Wang Li

Department of Biochemistry and Molecular Biology, Xinxiang Medical College, Xinxiang   453003, Henan Province, China

Zan Yu-xi, Lecturer, Department of Biochemistry and Molecular Biology, Xinxiang Medical College, Xinxiang   453003, Henan Province, China
zanyuxi@xxmu.edu.cn

Correspondence to: Wang Tian-yun, Doctor, Associate professor, Department of Biochemistry and Molecular Biology, Xinxiang Medical College, Xinxiang   453003, Henan Province, China
wty@xxmu.edu.cn

Supported by: the National Natural Science Foundation of China, No. 30470030*; the Natural Science Foundation of Henan Province, No. 0511042300*; the Tackle Key Program in Science and Technology of Henan Province, No. 0624410041*

Received: 2007-11-24
Accepted: 2008-04-01

Abstract
BACKGROUND: Matrix attachment region (MAR), a DNA sequence, is still bound to the nuclear matrices after chromatin digested with restriction endonuclease, not only affects expression of endogenous gene, but also overcames transgenic silence and improves transcription and expression of exogenous gene.
OBJECTIVE: To investigate the influence of β-interferon MAR of CHO cells on the transgenic expression of chloramphenicol acetyltransferase (CAT).
DESIGN, TIME AND SETTING: The opening experiment was performed at the Department of Biochemistry and Molecular Biology, 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 G418 screening markers were constructed by this laboratory.
METHODS: Human β-interferon MAR by PCR was digested with SacI/KpnI and BamHI/SalI, and was inserted into pCATG vector, which was propagated in Escherichia coli JM109, then extracted and purified followed by enzyme digestion analysis. Vector of CAT expression cassette and human β-interferon MAR by the two sides was successfully constructed, and christened as pCAT-MAR. Two methods were compared between CHO cells of pCATG transformation and CHO cells of pCATG-MAR transformation. After G418 selecting, genome DNA of cell lines of G418 was extracted, then primers for PCR to amplify the CAT target gene fragment was designed.
MAIN OUTCOME MEASURES: The activity of CAT was analyzed by ELISA method. It was also tested to see if the pCATG-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-MAR transformation was screened to have 17 strains of positive cell. Human β-interferon MAR could increase the CAT gene expression by 2.8 fold. The coefficient of variation of CHO cells of pCATG transformation was 2.065 0, and coefficient of variation of CHO cells of pCATG-MAR transformation was 0.813 1. Genome DNA of stable transformation cell lines was amplified by a fragment of 437 bp. The results confirmed the pCAT-MAR vector was stably integrated into genomic DNA.
CONCLUSION: Human β-interferon MAR can increase transgenic expression in CHO cells and decrease the transgenic expression variation in different transfected cells.

Zan YX, Wang TY, Zhang JH, Wang L.Regulatory effect of human beta-interferon matrix attachment region on transgene expression in CHO cells.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(29):5623-5626(China)
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