Construction of an EBNA-producing line of well-differentiated human hepatoma cells and of appropriate Epstein-Barr virus-based shuttle vectors.
Using cloned Epstein-Barr nuclear antigen 1 (EBNA) and oriP elements from the Epstein-Barr virus (EBV) in conjunction with liver-specific growth media, we have constructed an EBNA-producing line of well-differentiated human hepatoma cells (Hep-EBNA-2) and appropriate EBV-oriP vectors. These vectors, pBEDC1 and pBEUG1, were maintained as free extrachromosomal elements only in cells that expressed the trans-acting EBNA protein. They were readily rescued from transfected Hep-EBNA-2 cells upon transformation of recA- Escherichia coli with cellular low-Mr DNA. They are true shuttle vectors in that they can propagate as free closed circular elements in both human Hep-EBNA-2 cells and E. coli. Finally, we have demonstrated the vector capability of our shuttle system by inserting into the SV40 expression cassette of pBEUG1 a large full-length cDNA encoding coagulation factor VIII. Our data clearly show that EBV-oriP episomes are able to stably propagate in an hepatic background and that neither high levels of EBNA protein nor multiple copy episomes significantly interfere with the expression of the set of hepatic functions that have been analyzed. These results are discussed in terms of gene amplification and cloning of genes that program liver differentiation.
Lutfalla
G
G
Centre de Génétique Moléculaire du CNRS, Gif-sur-Yvette, France.
Armbruster
L
L
Dequin
S
S
Bertolotti
R
R
D000956:Antigens, Viral / Q000096:biosynthesis* / Q000235:genetics; D015139:Blotting, Southern; D006528:Carcinoma, Hepatocellular / Q000235:genetics* / Q000473:pathology; D003001:Cloning, Molecular; D003360:Cosmids; D004247:DNA; D019309:Epstein-Barr Virus Nuclear Antigens; D004926:Escherichia coli / Q000235:genetics; D005169:Factor VIII / Q000096:biosynthesis / Q000235:genetics; D005455:Fluorescent Antibody Technique; D005784:Gene Amplification; D005822:Genetic Vectors; D004854:Herpesvirus 4, Human / Q000235:genetics / Q000276:immunology*; D008099:Liver / Q000166:cytology; D008113:Liver Neoplasms / Q000235:genetics* / Q000473:pathology; D010957:Plasmids; D015183:Restriction Mapping; D014162:Transfection; D014407:Tumor Cells, Cultured / Q000378:metabolism*
D000956:Antigens, Viral; D019309:Epstein-Barr Virus Nuclear Antigens; D005169:Factor VIII; D004247:DNA