Gene replacement with one-sided homologous recombination.

Abstract

Homologous recombination is now routinely used in mammalian cells to replace endogenous chromosomal sequences with transferred DNA. Vectors for this purpose are traditionally constructed so that the replacement segment is flanked on both sides by DNA sequences which are identical to sequences in the chromosomal target gene. To test the importance of bilateral regions of homology, we measured recombination between transferred and chromosomal immunoglobulin genes when the transferred segment was homologous to the chromosomal gene only on the 3' side. In each of the four recombinants analyzed, the 5' junction was unique, suggesting that it was formed by nonhomologous, i.e., random or illegitimate, recombination. In two of the recombinants, the 3' junction was apparently formed by homologous recombination, while in the other two recombinants, the 3' junction as well as the 5' junction might have involved a nonhomologous crossover. As reported previously, we found that the frequency of gene targeting increases monotonically with the length of the region of homology. Our results also indicate that targeting with fragments bearing one-sided homology can be as efficient as with fragments with bilateral homology, provided that the overall length of homology is comparable. The frequency of these events suggests that the immunoglobulin locus is particularly susceptible to nonhomologous recombination. Vectors designed for one-sided homologous recombination might be advantageous for some applications in genetic engineering.Homologous recombination between transferred and chromosomal DNA provides a method of introducing predetermined changes into genes in their normal chromosomal location. The strategies which have been developed for replacing segments of the chromosomal genes of mammalian cells have been based on the requirements for recombination in microorganisms, and in the gene replacement vectors which have been used to date, the replacement segment has been flanked on both ends by homologous regions ranging in length from 0.1 to 5 kb (1,6,7,17,19,20). Here we describe recombination between transferred and chromosomal immunoglobulin genes in hybridoma cells when the replacement segment is bounded by homologous DNA on one side only. MATERIALS AND METHODSCell lines and plasmids. The Sp6, igm482, and igm692 cell lines, tissue culture methods, and plasmids containing the DNA fragments used for electroporation have been previously described (11). The pRCp.-E vector bears the 9.5-kb EcoRI C,u fragment from pR-Sp6 (14) ferred with a Pasteur pipette to medium in 96-well plates. These cells were expanded, and the plaque plucking was repeated. After several rounds of enrichment by this technique, the PFC were isolated and cloned by limiting dilution. In order to isolate independent recombinants, the cells were subdivided immediately after electroporation, and only one PFC was taken from each subculture.Analysis of recombinant hybridoma clones. The p. heavy chains of the recombinants were analyzed by Western immunoblots. The immu...

Introduction of double-strand breaks into the genome of mouse cells by expression of a rare-cutting endonuclease.

“…We have detected two major classes of gene-targeted clones, those derived from two-sided homologous recombination and those derived from homologous recombination on one side and nonhomologous recombination on the other side. Interestingly, such one-sided events have previously been observed in other mammalian gene targeting systems in the absence of a DSB at the target locus (4,11,21,57).…”

Theoretical mechanisms in targeted and random integration of transgene DNA

“…However, only the DSBR model is able to account for yeast transfection data. In terms integration at the other [1]. A further possibility, involving a variant form of DSBR has been suggested by Li et al [24].…”

Evidence for Biased Holliday Junction Cleavage and Mismatch Repair Directed by Junction Cuts during Double-Strand-Break Repair in Mammalian Cells

“…Hybridoma cell lines with unexpected C region fragments or those that have suffered a deletion of the endogenous C locus have been observed in previous gene targeting studies (4,25,31). Although they have not been fully characterized, these may represent cases where one arm of the vector has been degraded, forcing the cells to undergo one-sided recombination with the target locus or perhaps illegitimate recombination such as has been reported previously (4,7,21). In summary, of the 40 G418 r transformants initially identified by the PCR screening, 26 bore the correct gene structure required for determination of the crossover mechanism, and therefore these hybridoma cell lines were saved for further analysis.…”