A model for the mechanism of precise integration of a microinjected transgene

Abstract

A unique transgenic mouse line has undergone transgene integration in a very precise fashion. The phenotype displayed by mice of the line followed the predicted inheritance patterns for X-linked transgene insertion which has been confirmed. In order to investigate the mechanism of integration the DNA sequence of the transgene and cellular junctions have been determined. A comparison between wild type and transgenic mutant sequences at the site of insertion revealed that there was no loss or rearrangement of cellular DNA upon integration of the transgene. The cellular sequences at the transgene 5' and 3' joins are contiguous in the wild type. The integrant exists as a head to tail tandem dimer with minimal loss of sequence compared with the injected monomer. Analysis of the site of insertion has revealed a 5 bp homology between the 5' end of the transgene and the cellular sequences. In addition, adjacent to the site of insertion within the cellular sequences, there are several sequence motifs implicated in recombination events including a clustering of strong consensus sites of DNA topoisomerase type I and a region of homology to the human minisatellite consensus core sequence, the Escherichia coli Chi site and the meiotic recombination hotspot within the E beta gene of the murine major histocompatibility complex. This clustering of features is likely to have been factorial in the integrity of the insertion event. A model depicting the mechanism of this precise integration is proposed.

Illegitimate DNA integration in mammalian cells

“…Such findings are a recurrent theme: many other investigators have found Topo I consensus sequences, and other sequences such as Escherichia coli CHI element and minisatellite sequences, near transgene-chromosome junctions. 43,44,59,94,95 It has been demonstrated that integration of a circular bent DNA-containing vector preferentially occurs at the bent site, while bent DNA structures are often found at illegitimate recombination junctions and rearrangement breakpoints. 76 This suggests that such bent DNA sites in the genome (which are numerous) might be involved in integration.…”

“…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.…”

“…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). 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.…”

Efficient Integration of Short Interspersed Element-flanked Foreign DNA via Homologous Recombination

“…In fact, as described in our another report, the p(B1/2) 2 ␤geo-injected blastocyst embryos showed much higher levels of ␤-gal expression compared with the p␤geo-injected control embryos. 2 It is generally accepted that the frequency of random integration into a genome is highly affected by the sequences of transgene ends (26,37). In measuring the integration efficiency of SINE-flanked vectors by the microinjection experiment we excluded such potential bias by linearizing both control and SINE-tagged vectors with the ScaI restriction enzyme, which cuts both plasmids once within the ampicillin-resistant gene leaving the repetitive sequences positioned internally in the vectors.…”

Murine CASK Is Disrupted in a Sex-Linked Cleft Palate Mouse Mutant

“…Unusually for microinjected transgene integrations, the sequences on either side of the transgene insertion showed absolutely no rearrangements, nucleotide losses, or nucleotide additions compared to the wildtype locus and were found to be contiguous in wildtype murine DNA (McFarlane and Wilson, 1996). The genomic sequences immediately adjacent to the transgene insertion site were found to be unique and were used to map the disrupted locus by an interspecific backcross analysis (locus map nomenclature DXRib1/ DXPri1).…”