Foreign DNA introduced by calcium phosphate is integrated into repetitive DNA elements of the mouse L cell genome.

Abstract

We investigated the sites of integration of exogenous DNA fragments introduced by DNA-mediated gene transfer. Mouse Ltk-cells were transformed with the herpes simplex virus thymidine kinase gene and pBR322 DNA by the calcium phosphate precipitation method. Some of the integrated exogenous DNA sequences were recovered from the stable tk+ transformants in the form of plasmids that were capable of propagation in bacteria. Four plasmids derived from two cloned cell lines were analyzed in detail by nucleotide sequencing and hybridization techniques. These plasmids contained a total of seven cellular-exogenous DNA junctions. In all cases, there was no sequence homology between the exogenous and cellular DNA sequences adjacent to the joining sites, and no specific exogenous or cellular sequences occurred at the junctions. Rearrangement or deletion of Ltk-DNA was always associated with the integration of exogenous DNA. All of the assignable cellular sequences at the junctions were repetitive sequences. Two of these sequences were from the MIF-1 repetitive sequence family, and a third consisted of a 40-base pair simple copolymer of alternating deoxyadenosine-deoxythymidine. Our results suggest that repetitive sequences are relatively favorable sites for the integration of exogenous DNA.Homologous recombination of foreign DNA with the host genome is sufficiently frequent in procaryotes (33) and lower eucaryotes (18) that it is possible to confidently predict the integration site of the introduced DNA. Since such efficient "targeting" of DNA into homologous sequences has not yet been demonstrated in mammalian cells (41), we asked whether the foreign DNA is integrated randomly or at some preferred sites other than the homologous sequences. Although a number of workers have reported on the DNA sequences at the integration sites of viral DNAs that have been introduced into mammalian cells (3,11,37,39), there have been no reports of a similar analysis for the integration of nonviral DNAs. Even though it has been established that at the level of chromosome analysis foreign, nonviral DNAs are integrated randomly (36), there is no information on the DNA sequences at the integration sites of exogenous nonviral DNAs.Since the site of integration may be influenced by proteins encoded by the DNA in question (as appears to be the case for the genomes of some eucaryotic viruses [34]) as opposed to DNA which relies solely on the cellular machinery for its integration, we examined integration sites resulting from the transfer of purified gene fragments and procaryotic DNA. Following transformation of mouse Ltk-cells with a herpes simplex virus thymidine kinase gene (tk) fragment and pBR322 DNA, individual colonies of stable tk+ transformants were isolated and expanded. A plasmid rescue technique was used to recover the integrated exogenous DNA and flanking cellular sequences from the genomic DNA of these cell lines (1, 2). We analyzed the sequences encompassing seven cellular-exogenous DNA junctions. MATERIALS AND METHODSDNA-mediated ge...

Illegitimate DNA integration in mammalian cells

“…This mechanism has been suggested by numerous investigators, and this model receives the most support from the reviewed data. 42,58 According to this model, a genomic lesion, either single-or doublestranded, would attract repair factors. Any DNA with free ends, which happened to be in the vicinity or be recruited to the lesion by repair factors, could become part of the 'healing' process and be integrated (see Figure 3).…”

“…44 However, more complex insertion patterns are frequently observed (see Figure 2b). The recipient genomic locus can undergo extensive physical rearrangements at the site of integration, 45,50,51,58,59 including deletions (as much as tens of kb), duplications, and translocations, among others. 43,52,60 Another consequence is that the integrated material can interrupt coding sequences.…”

Acquisition of telomere repeat sequences by transfected DNA integrated at the site of a chromosome break.

“…In rodent cells, integration often involves * Corresponding author. large amounts of tandemly arranged DNA (35,38) and has been reported to be accompanied by large rearrangements in the surrounding cell DNA (19,38). However, the amount of DNA integrated into human cells is generally much less (9,17,22,23,29), and integration commonly occurs without major rearrangements in the cell DNA (33).…”

“…DNA transfected into mammalian cells most often integrates at random locations in the genome (19,33,35,38), although a small fraction can integrate into homologous sequences (16,37). In rodent cells, integration often involves * Corresponding author.…”

High-Frequency Illegitimate Integration of Transfected DNA at Preintegrated Target Sites in a Mammalian Genome

“…Mammalian cells integrate foreign DNA widely throughout the genome in a process often referred to as random integration (27,58). The integration of DNA into the mammalian genome is efficient, with up to 20% of cells integrating microinjected DNA (10).…”