Transfected DNA is mutated in monkey, mouse, and human cells.

Abstract

Papovavirus-based shuttle vectors containing the bacterial lacI gene were used to show that a mutation frequency in the range of 1% occurs in lacI when such vectors are transfected into COS7 and CV-1 simian cells, NIH 3T3, 3T6, L, and C127 mouse cells, and human 293 and HeLa cells. This frequency is approximately four orders of magnitude higher than the spontaneous mutation frequency in either mammalian or bacterial cells. The mutations are predominantly base substitutions and deletions and also include insertions from the mammalian genome. Time course experiments argue that mutagenesis occurs soon after arrival of the DNA into the nucleus. However, replication of the vector is not required since mutations occur even when the vector lacks all viral sequences. The high mutation frequency appears to be the characteristic outcome of transfection of DNA into mammalian cells.Mammalian cells in culture readily take up DNA. Only a small portion of this transfected DNA reaches the nucleus, and yet a smaller fraction is stably incorporated into the chromosomes in a situation permitting its expression. Although the transfection process is poorly understood, transfection of DNA has become a tool of pivotal importance in the study of the molecular biology of mammalian cells. Application of the technique has facilitated study of topics such as the expression of manipulated genes and the identification of sequences with oncogenic potential. We have sought to use DNA transfection to study mutation in mammalian cells by incorporating an easily scored marker on a transfected vector. Previously, the lacd gene of Escherichia coli was used as the mutational target on simian virus 40 (SV40)-based vectors that could replicate in both COS7 simian cells and bacteria. We found that, after transfection by the DEAE-dextran technique and 2-day passage in COS7 cells, 1% of the plasmids rescued to E. coli contained mutations in lacI (3). A similar mutation frequency was detected in related vectors, using the galK marker (29). This strikingly high mutation frequency and the facility of the lacI system for detailed analysis of the mutations have led us to extend this approach by using different vectors and cell lines, to characterize further the mutational fate of transfected DNA.In this study, a series of vectors was introduced into a variety of mammalian cell lines to establish the generality of the high mutation frequency. A description of the host DNA that has in some cases become joined to the vectors is also included. The kinetics of mutagenesis were examined by performing time course experiments. To determine whether replication is required for mutagenesis, we introduced molecules lacking a viral origin of replication.Our observations argue that all transfected DNA, with or without any viral DNA, appears extremely susceptible to mutagenesis in mammalian cells. These findings are compatible with studies that indicate that cotransfected DNA species can become covalently joined, both by homologous recombination and by blunt-end ligation, a...

A phage integrase directs efficient site-specific integration in human cells

“…The remaining colonies contained plasmids that carried large rearrangements that disrupted lacZ. The low-frequency rearrangement of transfected plasmids has been observed with all plasmids, with and without integrase and att sites, and can be attributed to transfection-associated mutation of newly introduced DNA (15). This result indicates that the C31 integrase is active in mammalian cells and efficiently carries out the integration reaction when transiently introduced with its att sites.…”

Theoretical mechanisms in targeted and random integration of transgene DNA

“…Of the DNA that survives this journey, a substantial proportion sustains some degree of endonucleolytic or exonucleolytic damage. In contrast, virtually no damage occurs to DNA delivered directly into the nucleus by microinjection [22,47].…”

Spectrum of spontaneous mutation at the APRT locus of Chinese hamster ovary cells: an analysis at the DNA sequence level.

“…11). Although the use of shuttle vectors facilitates the recovery and sequence analysis of mutations, this experimental approach has several drawbacks, often including an extraordinarily high mutation frequency and a high percentage of deletions and rearrangements among the recovered plasmids (12)(13)(14)(15)(16). This is true even under conditions in which the shuttle vector has been incorporated into the chromosome, although some inserts appear to approach the stability expected for natural genes (17,18).…”