Article

Different matrix attachment regions flanking a transgene effectively enhance gene expression in stably transfected Chinese hamster ovary cells.

Department of Biochemistry and Molecular Biology, Xinxiang Medical University, Henan, People's Republic of China.
Gene (impact factor: 2.34). 03/2012; 500(1):59-62. DOI:10.1016/j.gene.2012.03.049 pp.59-62
Source: PubMed

ABSTRACT Numerous matrix attachment regions (MARs) have been used to improve transgene expression in genetic engineering, but an efficient and stable expression vector is lacking. In the present study, a vector named pCCF containing chloramphenicol acetyltransferase (CAT) reporter gene cassettes was constructed. The cassettes were flanked by a β-interferon MAR at the 5' upstream of the reporter gene cassettes, and a β-globin MAR at the 3' site. After transfecting pCCF into Chinese hamster ovary cells, the expression level of the CAT gene with a MAR was effectively increased to about 4.5-fold higher than that transfected with pCAM (containing two β-globin MARs flanking the expression cassette), and to 46.4-fold higher than that transfected with the control plasmid pCAG (without MARs). Quantitative reverse transcription polymerase chain reaction and the 2(-ΔΔCt) method were used to analyze the CAT gene relative copy numbers. The expression levels were found to be not directly proportional to the gene copy numbers when MAR elements from different sources were used. However, the presence of MARs improved the transgene copy numbers.

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    Article: Efficient recombinant production in mammalian cells using a novel IR/MAR gene amplification method.
    Yoshio Araki, Tetsuro Hamafuji, Chiemi Noguchi, Noriaki Shimizu
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    ABSTRACT: We previously found that plasmids bearing a mammalian replication initiation region (IR) and a nuclear matrix attachment region (MAR) efficiently initiate gene amplification and spontaneously increase their copy numbers in animal cells. In this study, this novel method was applied to the establishment of cells with high recombinant antibody production. The level of recombinant antibody expression was tightly correlated with the efficiency of plasmid amplification and the cytogenetic appearance of the amplified genes, and was strongly dependent on cell type. By using a widely used cell line for industrial protein production, CHO DG44, clones expressing very high levels of antibody were easily obtained. High-producer clones stably expressed the antibody over several months without eliciting changes in both the protein expression level and the cytogenetic appearance of the amplified genes. The integrity and reactivity of the protein produced by this method was fine. In serum-free suspension culture, the specific protein production rate in high-density cultures was 29.4 pg/cell/day. In conclusion, the IR/MAR gene amplification method is a novel and efficient platform for recombinant antibody production in mammalian cells, which rapidly and easily enables the establishment of stable high-producer cell clone.
    PLoS ONE 01/2012; 7(7):e41787. · 4.09 Impact Factor
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Keywords

5' upstream
 
Chinese hamster ovary cells
 
chloramphenicol acetyltransferase
 
control plasmid pCAG
 
different sources
 
expression cassette
 
expression levels
 
gene copy numbers
 
genetic engineering
 
MAR elements
 
MARs
 
Numerous matrix attachment regions
 
reporter gene cassettes
 
stable expression vector
 
transfecting pCCF
 
transgene copy numbers
 
transgene expression
 
β-globin MAR
 
β-globin MARs flanking
 
β-interferon MAR
 

Fang Wang