Cryptic promoter activity within the backbone of a plasmid commonly used to prepare promoter/reporter gene constructs

Edward Rosfjord, Kimberly Lamb, Angie Rizzino

Research output: Contribution to journalArticlepeer-review

24 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)477-481
Number of pages5
JournalIn Vitro Cellular & Developmental Biology - Animal
Volume30
Issue number7
DOIs
StatePublished - Jul 1994

Keywords

  • embryonal carcinoma cells
  • pBLCAT3
  • promoter reporter gene constructs
  • transient expression assays

ASJC Scopus subject areas

  • Developmental Biology
  • Cell Biology

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