Derepression of a mouse α-fetoprotein expression vector in COS-1 cells by amplification of specific cis-acting sequences of the AFP promoter

J. P. Rabek, P. R. Hoyt, D. E. Zhang, M. G. Izban, J. Papaconstantinou

Research output: Contribution to journalArticle

6 Scopus citations

Abstract

The existence of trans-acting regulatory factors has been demonstrated by in vivo competition with cis-acting sequences from both viral and eukaryotic genomes. Plasmids containing a functional SV40 origin of replication when transfected into permissive SV40 T-antigen producing COS-1 cells will amplify to high copy numbers (5,000 to 10,000) without inflicting toxic effects upon the host cell. This amplification vector (pSVori) has been used to amplify cis-acting regulatory elements which can act as competitors for positive and negative trans-acting factors in vivo. Using this amplification system we conducted experiments to determine whether amplification of α-fetoprotein (AFP) and albumin cis-acting promoter sequences could activate a corresponding co-transfected AFP-promoter-CAT or Alb-promoter-CAT expression vector in COS-1 cells. We used pMoMLV(-1009)AFPcat, or p(-308)Albcat-MoMLV as reporter genes and pSVori to amplify specific promoter sequences of the AFP or albumin promoter. Our experiments indicated that amplification of a region from - 53 to - 202 of the AFP promoter resulted in the activation of the pMoMLV(-1009)AFPcat and p( - 308)Albcat-MoMLV expression vectors in COS-1 cells. Surprisingly, amplification of the albumin promoter sequences failed to activate either the pMoMLV(-1009)AFPcat or p(-308)Albcat-MoMLV plasmids.

Original languageEnglish (US)
Pages (from-to)6677-6682
Number of pages6
JournalNucleic acids research
Volume18
Issue number22
DOIs
StatePublished - Nov 25 1990

ASJC Scopus subject areas

  • Genetics

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