Inefficiency of expression of luciferase reporter from transfected murine leukaemia proviral DNA may be partially overcome by providing a strong polyadenylation signal.
HepG2 human hepatoma cells were transfected with the luciferase reporter gene, linked with a liver-specific enhancer plus a minimal promoter, contained within either pBR/pUC or Moloney murine leukaemia virus (MMLV) proviral plasmid contexts. Reporter expression from the proviral plasmid was decreased 10- to 20-fold, regardless of whether or not the orientation within the proviral DNA was appropriate for the use of the poly(A) signal in the 3' long terminal repeat (LTR). Efficient reporter expression was restored when the proviral transcription unit was provided with a simian virus 40 poly(A) signal. These results imply that the MMLV LTR poly(A) signal is inefficient. Therefore, strategies to maximize expression of internal transcription units from retroviral vectors should include the provision of an efficient (unidirectional) poly(A) signal (its requiring insertion in the reverse orientation to that of viral transcription).
Maxwell
I H
IH
Department of Medicine, University of Colorado Health Sciences Center, Denver 80262.
Brown
J L
JL
Maxwell
F
F
D006528:Carcinoma, Hepatocellular; D004279:DNA, Viral / Q000737:chemistry; D004742:Enhancer Elements, Genetic; D015967:Gene Expression Regulation, Viral; D006801:Humans; D008113:Liver Neoplasms; D008156:Luciferases / Q000235:genetics*; D008979:Moloney murine leukemia virus / Q000201:enzymology / Q000235:genetics*; D010957:Plasmids; D011061:Poly A / Q000235:genetics; D011401:Promoter Regions, Genetic; D011533:Proviruses / Q000235:genetics*; D014162:Transfection; D014407:Tumor Cells, Cultured
D004279:DNA, Viral; D011061:Poly A; D008156:Luciferases