High-frequency transformation of human repair-deficient cell lines by an Epstein-Barr virus-based cDNA expression vector.
We constructed a human cDNA expression vector by combining an episomal Epstein-Barr virus (EBV) vector with the expression cassette from the transient-expression vector, pCDM8. This new vector, designated pEBS7, exhibited high-level expression of reporter genes in normal and repair-deficient xeroderma pigmentosum cell lines. Reconstruction experiments indicated that marker genes diluted to a frequency of 10(-5) can be rescued on a single transfection dish. Moreover, derivative cell lines that constitutively express the gene encoding EBV nuclear antigen 1 exhibited a tenfold enhancement in the frequency of rescue of marker genes. The feasibility of preparing large-scale directional or nondirectional cDNA libraries in pEBS7 was demonstrated and reconstruction experiments indicated that marker genes could be rescued from either library with equal efficiency. These results establish a high-efficiency system for the isolation of genes by direct phenotypic selection in human mutant cell lines.
Peterson
C
C
Department of Molecular Genetics, University of Texas M.D. Anderson Cancer Center, Houston 77030.
Legerski
R
R
D000956:Antigens, Viral / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D004260:DNA Repair / Q000235:genetics*; D019309:Epstein-Barr Virus Nuclear Antigens; D015870:Gene Expression / Q000235:genetics; D015723:Gene Library; D005822:Genetic Vectors / Q000235:genetics*; D004854:Herpesvirus 4, Human / Q000235:genetics* / Q000276:immunology; D006801:Humans; D008969:Molecular Sequence Data; D010641:Phenotype; D010957:Plasmids / Q000235:genetics; D014162:Transfection; D014983:Xeroderma Pigmentosum / Q000235:genetics*
D000956:Antigens, Viral; D019309:Epstein-Barr Virus Nuclear Antigens