Efficiency of insertion versus replacement vector targeting varies at different chromosomal loci.

Abstract

We have analyzed the targeting frequencies and recombination products generated with (1,(12)(13)(14)39). Vectors with a DSB in the homologous counterpart of the chromosomal sequences have been termed insertion vectors and usually recombine through a single reciprocal pathway (25).The DSB repair (DSBR) model, originally proposed to explain the repair of breaks in DNA, has been invoked to explain the elevation of targeting frequencies in S. cerevisiae observed by the provision of a DSB in the homologous sequences (24,27,35). Although this model was originally developed with S. cerevisiae, it is supported by data for vector-chromosome recombination in mammalian cells (12,14,39 (13, 41). These insertion-like products suggest a preference for the integration of replacement vectors via an insertion pathway after end-end joining, possibly via a mechanism similar to that described by the DSBR model (13, 38).The presence of free ends of a vector, either homologous or heterologous, may be important for directing specific targeted integration pathways which in turn may affect both the targeting frequencies and the integration products. Previously, it has been shown that at the Aprt locus in CHO cells and at an integrated simian virus 40 genome in COS1 cells, when a DSB is made in the homologous sequences of the vector, the gene targeting frequency increases relative to the frequencies observed when the DSB is made outside these elements (1,14). However, at the immunoglobulin heavy-chain constant region in hybridoma cells, replacement and insertion vectors target at about the same frequency (15). We have reported previously that at the hprt locus in embryonic stem (ES) cells insertion vectors target more efficiently than replacement vectors (11-13), yet the Capecchi laboratory (9, 37) has reported that the two vector types target at about the same efficiency.Free DNA ends are also important for extrachromosomal homologous recombination in mammalian cells. DSBs in the homologous sequences in either of the substrates involved in the exchange increase the recombination frequency (5, 6, 17). As predicted by the DSBR pathway, both insertion and replacement products are observed. However, other reports have shown that intermolecular extrachromosomal homologous recombination is increased if both introduced DNAs are linearized within or just close to the homologous sequences. To explain this observation, the single-strand annealing (SSA) model is invoked. In the SSA model, the breaks appear to be substrates for exonuclease or helicase activity which generates complementary single-stranded DNA ends. These homologous single-stranded regions can anneal to each other and result in a replacement product (2,19,30,40). The location of the break does not need to be flanked by homology as long as singlestrand homology is exposed after exonuclease digestion or 8385

A schizophrenia-related sensorimotor deficit links α3-containing GABA A receptors to a dopamine hyperfunction

“…1A). Homologous recombination with insertion vectors requires a single reciprocal recombination, whereas homologous recombination with replacement vectors requires a double reciprocal recombination, and it has been shown that, at least in some cases, insertion vectors of the type used here may have significantly higher targeting frequencies than do replacement vectors (20). Insertion vector IV-3KO was linearized at a SpeI site within the 5Ј homology and completely integrated into the ES cell genome (targeted allele 1), which thus contains a duplication of exon 4, which is predicted to shift the reading frame and, thus, prevent formation of the ␣3-subunit protein.…”

Transgenic mice with targeted inactivation of the Col2 alpha 1 gene for collagen II develop a skeleton with membranous and periosteal bone but no endochondral bone.

“…About 15 kb of the clone was sequenced. The clone was then used to prepare a construct of the mouse Col2al gene designed for homologous recombination in embryonic stem (ES) cells (Capecchi 1989;Mortensen 1993;Hasty et al 1994). The construct extended from intron 31 to exon 43 of the Col2al gene (Fig.…”

“…1A). A neomycin-resistance gene was inserted into exon 35, and the thymidine kinase gene from herpes simplex virus was ligated to the 5' end of the construct (Mortensen 1993;Hasty et al 1994). The neomycin-resistance gene introduced an additional BamHI site into the targeted locus (Fig.…”

Preparation of Animals with a High Degree of Chimerism by One-Step Coculture of Embryonic Stem Cells and Preimplantation Embryos

“…In general, a positive-negative selection gene-targeting cassette is prepared in which an exon of the target gene is interrupted by the gene for neomycin resistance (5,9,12). A herpes simplex virus thymidine kinase (HSV TK) gene is fused at either one end or both ends of the genomic sequence as a negative selection marker.…”

“…A herpes simplex virus thymidine kinase (HSV TK) gene is fused at either one end or both ends of the genomic sequence as a negative selection marker. The targeting cassette is introduced into ES cells by electroporation, and the cells in which the endogenous gene is disrupted by homologous recombination are selected with G418 and gancyclovir or FIAU (5,12).…”