Establishment of an oriP/EBNA1-based episomal vector transcribing human genomic beta-globin in cultured murine fibroblasts.
A novel oriP/EBNA1-based episomal vector has been constructed that persists episomally in cultured murine fibroblasts. The vector, pBH148, is equipped with the entire 185-kb human beta-globin gene locus. After amplification in bacteria, column-purified episomal pBH148 was transfected into both cultured EBNA1-expressing human D98/Raji positive control fusion cells (DRpBH148) and cultured EBNA1-negative murine fibroblast cells (A9pBH148). Cell cultures were maintained concurrently with and without hygromycin selection for a period of 3 months. We show long-term stable episome maintenance of the full-size 200-kb circular double-stranded pBH148 in both the DRpBH148 cultures and the A9pBH148 cultures, regardless of selective pressure by agarose gel electrophoresis and Southern blot. EBNA1 transgene was detected by PCR in all transfected cultures. In addition, we were able to detect correctly spliced human beta-globin mRNA by RT-PCR in all transfected late-passage DRpBH148 and A9pBH148 cell cultures. These findings illustrate that this oriP/EBNA1-based episomal vector is stable in a previously nonpermissive murine cell line and is a potential vector for human gene therapy.
Black
J
J
Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, USA.
Vos
J-M
JM
D000818:Animals; D015139:Blotting, Southern; D002460:Cell Line; D019309:Epstein-Barr Virus Nuclear Antigens / Q000235:genetics*; D005347:Fibroblasts / Q000378:metabolism; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000008:administration & dosage / Q000235:genetics*; D005914:Globins / Q000032:analysis / Q000235:genetics*; D006801:Humans; D051379:Mice; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014158:Transcription, Genetic
D019309:Epstein-Barr Virus Nuclear Antigens; D005914:Globins; C417029:EBV-encoded nuclear antigen 1