Spontaneous mutations in the human gene for p53 in recombinant adenovirus during multiple passages in human embryonic kidney 293 cells.
Infectious recombinant adenovirus (rAd) is usually produced in human embryonic kidney 293 cells that harbor the E1 gene and rAd has been shown to be an efficient tool for gene transfer both in vivo and in vitro. It also has considerable potential in human gene therapy. However, rates of spontaneous mutations in genes introduced into host cells after multiple passages remain to be clarified. We have characterized the spontaneous mutation of genomes derived from human adenovirus type 5 (Ad5) and of human p53-rAd during multiple passages by two different methods, namely, a plaque assay and a molecular cloning assay, with subsequent direct nucleotide sequencing. Using the plaque assay, we found no mutations in the E1A and p53 genes derived from infectious Ad5 and p53-rAd, respectively. By contrast, we found spontaneous mutations in the E1A gene of Ad5, with a mutation rate of 9.28 x 10(-8) per base pair per plaque, in the molecular cloning assay. The rate of mutation of the p53 gene of p53-rAd, as determined by the molecular cloning assay, ranged from 1.50 x 10(-7) to 3.25 x 10(-7) per base pair per passage. The mutations in the p53 gene of p53-rAd were localized mainly in the transcriptional activation domain, the SH3 domain, and the regulation domain and they were rarely found in the DNA-binding domain, which is a major site for mutations in human cancers. Our results indicate that multiple passages can generate a heterogeneous population of p53-rAd and that the molecular cloning assay is an efficient technique with which to search for mutations in the genome of p53-rAd that cannot be detected by a plaque assay.
Ugai
Hideyo
H
Gene Engineering Division, Department of Biological Systems, BioResource Center, RIKEN - The Institute of Physical and Chemical Research, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan.
Suzuki
Erika
E
Inabe
Kumiko
K
Murata
Takehide
T
Hamada
Hirofumi
H
Yokoyama
Kazunari K
KK
D000256:Adenoviridae / Q000235:genetics* / Q000254:growth & development; D002460:Cell Line; D003001:Cloning, Molecular; D004274:DNA, Recombinant / Q000235:genetics; D005787:Gene Frequency; D016158:Genes, p53; D005822:Genetic Vectors; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000196:embryology; D009154:Mutation; D010948:Viral Plaque Assay
D004274:DNA, Recombinant