Cryptic promoter activity within the backbone of a plasmid commonly used to prepare promoter/reporter gene constructs.
This report demonstrates that the plasmids, pBLCAT2 and pBLCAT3, which are used widely for the preparation of promoter reporter gene constructs, exhibit cryptic promoter activity when expressed in embryonal carcinoma (EC) cells and their differentiated cells. The promoterless plasmid pBLCAT3 is used widely because it has two multiple cloning sites. We demonstrate that the activity of the cryptic promoter present in pBLCAT3 is increased dramatically by an enhancerlike region of the murine k-FGF gene. However, the basal cryptic promoter activity and the enhanced cryptic promoter activity can be silenced effectively by the insertion of three tandemly arranged polyadenylation sequences. To characterize the influence of the cryptic promoter in pBLCAT3, we tested its effects on two promoters. Our findings suggest that the cryptic promoter increases by several fold the expression of the reporter gene in pBLCAT2, which contains the thymidine kinase promoter. In contrast, the cryptic promoter present in pBLCAT3 does not seem to influence the expression of the k-FGF promoter. Last, we observed cryptic promoter activity when pBLCAT3 was expressed transiently in EC-differentiated cells. Together, our findings argue that transcription silencing sequences should be used when examining weak promoters in these plasmids, especially in combination with enhancers.
Eppley Institute for Research in Cancer and Allied Diseases, University Nebraska Medical Center, Omaha 68198-6805.
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Rosfjord
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Lamb
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Rizzino
D000818:Animals; D018236:Carcinoma, Embryonal / Q000235:genetics; D004742:Enhancer Elements, Genetic / Q000235:genetics; D051519:Fibroblast Growth Factor 4; D005346:Fibroblast Growth Factors / Q000235:genetics; D017930:Genes, Reporter / Q000235:genetics*; D051379:Mice; D010957:Plasmids / Q000235:genetics*; D011061:Poly A / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011518:Proto-Oncogene Proteins / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
C496446:Fgf4 protein, mouse; D051519:Fibroblast Growth Factor 4; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D011061:Poly A; D005346:Fibroblast Growth Factors