Patterns of integration of DNA microinjected into cultured mammalian cells: evidence for homologous recombination between injected plasmid DNA molecules.

Abstract

We examined the fate of DNA microinjected into nuclei of cultured mammalian cells. The sequence composition and the physical form of the vector carrying the selectable gene affected the efficiency of DNA-mediated transformation. Introduction of sequences near the simian virus 40 origin of DNA replication or in the long terminal repeat of avian sarcoma provirus into a recombinant plasmid containing the herpes simplex virus thymidine kinase gene (pBR322/HSV-tk) enhanced the frequency of transformation of LMtk-and RAT-2tk-cells to the TK+ phenotype 20-to 40-fold. In cells receiving injections of only a few plasmid DNA molecules, the transformation frequency was 40-fold higher after injection of linear molecules than after injection of supercoiled molecules. By controlling the number of gene copies injected into a recipient cell, we could obtain transformants containing a single copy or as many as 50 to 100 copies of the selectable gene. Multiple copies of the transforming gene were not scattered throughout the host genome but were integrated as a concatemer at one or a very few sites in the host chromosome. Independent transformants contained the donated genes in different chromosomes. The orientation of the gene copies within the concatemer was not random; rather, the copies were organized as tandem head-to-tail arrays. By analyzing transformants obtained by coinjecting two vectors which were identical except that in one a portion of the vector was inverted, we were able to conclude that the head-to-tail concatemers were generated predominantly by homologous recombination. Surprisingly, these head-to-tail concatemers were found in transformants obtained by

Illegitimate DNA integration in mammalian cells

“…56,70 Many studies on SV40 transformed cell lines reported sustained instability involving the SV40 sequences and flanking cellular DNA upon continued culturing, 56,[71][72][73] suggesting that integrated structures are often unstable immediately after integration, and that this instability can persist in the absence of selection. While in some cases, tandem arrays of transgenes remain stable following integration, 41 other groups have observed homologous recombination between copies within the array, which lead to changes in structure, expression, and transgene copy number 74,75 (see also our unpublished results).…”

“…In some cases, however, instability appears to be restricted to the time of integration. Many studies carried out in cultured cells 41,47,48,53 and transgenic mouse lines 43 have shown that while vectors can be physically rearranged initially, expansion of cell or mouse lines reveals no further rearrangements. This leads to the conclusion that the deletions and rearrangements observed are sometimes early events in the integration process.…”

“…It appears that exogenous DNA generally integrates at one, or very few, site(s) in the recipient genome, but often in multiple copies showing the presence of 1-6 bp of microhomology or slight degrada- Illegitimate DNA integration H Würtele et al tion or addition of nucleotides at transgene-transgene junctions. [41][42][43][44][45][46][47] The NHEJ activities capable of circularizing and concatemerizing extrachromosomal DNA have been suggested to account for randomly oriented transgene arrays, while the frequent appearance of tandem arrays comprised of direct repeats is best explained by homologous recombination between circular and linear molecules before or during the integration process. 41,42 The organization and quantity of integrated DNA appear to vary according to cell type.…”

“…[41][42][43][44][45][46][47] The NHEJ activities capable of circularizing and concatemerizing extrachromosomal DNA have been suggested to account for randomly oriented transgene arrays, while the frequent appearance of tandem arrays comprised of direct repeats is best explained by homologous recombination between circular and linear molecules before or during the integration process. 41,42 The organization and quantity of integrated DNA appear to vary according to cell type. A comparison of integrated transgenes between different cell lines suggested that the degree of genetic instability may be reflected in more complex integration patterns: 48 'normal' cell lines show simpler integration patterns than transformed ones.…”

“…[53][54][55][56] Finally, the transfection method can have an effect on the amount, and resulting form, of DNA integrating into a recipient genome. While calcium phosphate precipitation and microinjection result in relatively large amounts of DNA integrated in mouse cells, 39,41 electroporation shows a strong tendency for low copy numbers of integrated foreign DNA, and usually not in a standard head-to-tail array, but instead in a randomly arrayed structure. 57 The recipient chromosomal locus is often found modified after an integration event Some integration events are relatively 'harmless' to the integrity of a genomic site and can sometimes leave the recipient locus completely unchanged (apart from the inclusion of the new sequence).…”