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Transient expression in mammalian cells of the bacterial reporter gene encoding mercuric reductase: effects of
G Veldhuisen1, M Van Dijk, J Meijer
1TNO Medical Biological Laboratory, Rijswijk, The Netherlands.
Abstract:
The effect of several transcriptional regulatory elements on gene expression in mammalian cells was investigated. As a reporter gene we have used the bacterial gene merA coding for the enzyme mercuric reductase. Several plasmids were constructed with different promoter/enhancer sequences (pSV/E, pSV/L, pMT, pRSV or pAd) at the 5' end and different splicing (small intron of the T antigen of SV40 or the second intron of the rabbit beta-globin gene) and/or polyadenylation signals (AEn, ALn or AR beta Gn) at the 3' end of the merA gene. Expression was measured in five different mammalian cell lines. In COS cells the highest level of expression is obtained with pSV/L and the lowest level with pSV/E. In HeLa, CV-1, Ltk-, and CHO cells merA expression is relatively high, under control of pRSV and pMT and relatively low under control of pSV/L and pAd. The introns studied have a negative effect on the expression of merA. The presence of a polyadenylation signal downstream from the gene is essential for its expression. The three different polyadenylation signals studied give a similar stimulatory effect on the level of expression of the merA gene.
Insights
Investigating transcriptional regulatory elements, this study found that polyadenylation signals are essential for gene expression in mammalian cells. Introns negatively impacted expression, while specific promoters influenced reporter gene levels differently across cell lines.
Area of Science:
- Molecular Biology
- Gene Regulation
- Mammalian Cell Expression Systems
Background:
- Understanding transcriptional regulatory elements is crucial for controlling gene expression in mammalian cells.
- The bacterial merA gene, encoding mercuric reductase, was utilized as a reporter gene to assess regulatory element function.
Purpose of the Study:
- To investigate the impact of various promoter/enhancer sequences, splicing signals (introns), and polyadenylation signals on reporter gene expression.
- To compare the efficacy of different regulatory elements across diverse mammalian cell lines.
Main Methods:
- Construction of plasmids containing the merA reporter gene with varied 5' promoter/enhancer sequences and 3' splicing/polyadenylation signals.
- Quantification of merA gene expression in five distinct mammalian cell lines (COS, HeLa, CV-1, Ltk-, CHO).
Main Results:
- Promoter activity varied significantly, with pSV/L showing highest expression in COS cells and pRSV/pMT yielding high expression in other cell lines.
- Introns generally had a negative effect on merA expression.
- Polyadenylation signals were essential for gene expression, with all three tested signals showing a similar stimulatory effect.
Conclusions:
- Specific promoter and enhancer sequences differentially regulate gene expression in mammalian cells.
- Polyadenylation signals are critical for enabling gene expression, whereas introns can impede it.
- The choice of regulatory elements must be optimized based on the specific mammalian cell line used for expression.