Analysis of gene targeting and intrachromosomal homologous recombination stimulated by genomic double-strand breaks in mouse embryonic stem cells

Abstract

To investigate the effects of in vivo genomic DNA double-strand breaks on the efficiency and mechanisms of gene targeting in mouse embryonic stem cells, we have used a series of insertion and replacement vectors carrying two, one, or no genomic sites for the rare-cutting endonuclease I-SceI. These vectors were introduced into the hypoxanthine phosphoribosyltransferase (hprt) gene to produce substrates for gene-targeting (plasmid-to-chromosome) or intrachromosomal (direct repeat) homologous recombination. Recombination at the hprt locus is markedly increased following transfection with an I-SceI expression plasmid and a homologous donor plasmid (if needed). The frequency of gene targeting in clones with an I-SceI site attains a value of 1%, 5,000-fold higher than that in clones with no I-SceI site. The use of silent restriction site polymorphisms indicates that the frequencies with which donor plasmid sequences replace the target chromosomal sequences decrease with distance from the genomic break site. The frequency of intrachromosomal recombination reaches a value of 3.1%, 120-fold higher than background spontaneous recombination. Because palindromic insertions were used as polymorphic markers, a significant number of recombinants exhibit distinct genotypic sectoring among daughter cells from a single clone, suggesting the existence of heteroduplex DNA in the original recombination product.

FIG. 1

Genomic and plasmid restriction maps of hprt region. (A) The genomic region of hprt containing exons 2 and 3. (B) The 5.5-kb fragment of hprt used in these experiments (indicated by the heavy solid line in the genomic map) was modified at five sites to produce plasmid GPD384. Sites 1, 2, 3, 4, and 5 are approximately 300 bp, 1.3 kbp, 2.6 kbp, 4.2 kbp, and 5.0 kbp downstream of the XmnI site at the 5′ end of the 5.5-kb fragment, respectively. Restriction sites indicated are BamHI (Bam), BglII (Bgl), EcoRI (Eco), EcoRV (R5), HindIII (H3), NotI (Not), PstI (Pst), PvuII (Pvu), SalI (Sal), XbaI (Xba), XhoI (Xho), and XmnI (Xmn).

FIG. 2

Production of replacement series of hprt knockout clones. (A) Diagram of homologous integration into the genome by a BamHI/SalI-linearized plasmid(s) carrying the PGKneo G418^R marker to produce a set of hprt knockout clones varying only in the location, number, and orientation of the I-SceI-cut site(s). All possible HindIII/I-SceI digestion fragments, as detected by Southern blotting, are indicated by arrows. Restriction enzyme abbreviations are as explained for Fig. 1. (B) Southern blots of genomic DNA digested with HindIII and I-SceI from clones identified as having correct genomic integration structures were probed with hprt exons 2 and 3. Fragments containing only PGKneo or plasmid backbone-derived sequences are not detected.

FIG. 3

Production of insertion series of hprt knockout clones. (A) Diagram of homologous integration into the genome by an XbaI-linearized plasmid(s) carrying the PGKneo G418^R marker to produce a set of hprt knockout clones varying only in the location, number, and orientation of the I-SceI cut site(s). All possible HindIII/I-SceI digestion fragments, as detected by Southern blotting, are indicated by arrows. Restriction enzyme abbreviations are as explained for Fig. 1. (B) Southern blots of genomic DNA digested with HindIII and I-SceI from clones identified as having correct genomic integration structures were probed with hprt exons 2 and 3. Fragments containing only PGKneo or plasmid backbone-derived sequences are not detected.

FIG. 4

Targeting frequencies and genotypes of HAT^R recombinants from the replacement clones. (A) Targeting frequencies for replacement clones by calcium phosphate transfection with pPGK3XnlsI-SceI and circular GPD384 (solid bars), electroporation with pPGK3XnlsI-SceI and circular GPD384 (open bars), or electroporation with pPGK3XnlsI-SceI and linearized GPD384 (striped bars). (B) Overall gene conversion frequencies in HAT^R recombinant clones isolated after electroporation of clones 2B and 3A. Arrows indicate gene conversion from the DSBs to a particular site. (C) Summaries of individual genotype profiles for recombinants isolated from clones 2B and 3A. HAT^R clones are labeled to distinguish whether circular (C) or NotI-linearized (L) GPD384 was introduced as a donor substrate. Wild-type restriction sites, polymorphic restriction sites, and mixes of wild-type and polymorphic sites are represented by filled, open, and half-filled, half-open squares, respectively. Restriction enzyme abbreviations are as explained for Fig. 1.

FIG. 5

Targeting frequencies and genotypes of HAT^R recombinants from the insertion clones. (A) Targeting frequencies for insertion clones by calcium phosphate transfection (solid bars) or electroporation (open bars) with pPGK3XnlsI-SceI. (B) Overall gene conversion frequencies in HAT^R recombinant clones isolated after spontaneous recombination in clone 7B or electroporation of clones 8D and 9A. The percent clones showing gene conversion at a particular site is indicated. (C) Summaries of individual genotype profiles for recombinants isolated from clones 7B, 8D, and 9A. Because clone 9A had not incorporated a polymorphic marker at site 4, no conclusions regarding gene conversion at that location could be drawn. Wild-type restriction sites, polymorphic restriction sites, and mixes of wild-type and polymorphic sites are represented by filled, open, and half-filled, half-open squares, respectively. Restriction enzyme abbreviations are as explained for Fig. 1.

FIG. 6

Preservation of heteroduplex at palindromic restriction site insertion polymorphisms. (A) Heteroduplex formed between palindromic insertion polymorphism and wild-type restriction markers at sites 1 through 5 with no mismatched base pairs. (B) Demonstration of mixed genotypes of daughter strands after replication through a heteroduplex at site 1.

FIG. 7

Generation of trans heteroduplex in HAT^R clones containing mixed genotypes at sites 2 and 4. (A) The single-strand annealing model is diagrammed to explain the generation of simultaneous mixed genotypes at both sites 2 and 4 during intrachromosomal recombination. (B) The two possible outcomes of replicational resolution of unknown mixed genotypes at sites 2 and 4 are diagrammed. Resulting clones are identified on the basis of same-strand or opposite-strand orientation of polymorphisms. Wild-type restriction sites, polymorphic restriction sites, and mixes of wild-type and polymorphic sites are represented by filled, open, and half-filled, half-open rectangles, respectively.