Gene targeting at the Recql5 locus. (a) The targeting strategy to delete exon 4 of the Recql5 gene. The 19 exons encoding Recql5 beta are shown as boxes, and the sequences encoding the conserved helicase domain are indicated by black boxes. With this strategy, a correct targeting event replaces exon 4 with a LoxP-exon4-LoxP-PGKNeo-LoxP cassette, giving rise to the first Recql5 targeted allele, the M1 allele. Subsequently, a Cre-mediated deletion of sequences between the two loxP sites (open triangles) flanking the Neo cassette leads to a conditional Recql5 knockout allele, the M2 allele, in which exon 4 is flanked by a pair of loxP sites. Alternatively, a Cre-mediated deletion of both exon 4 and the Neo cassette results in a true Recql5 knockout allele, the M3 allele. Note the opposite directions of transcriptions for PGKNeo and Recql5. Neo, neomycin phosphotransferase gene; TK, thymidine kinase gene; X, XbaI; R, EcoR I. (b) Identifications of ES clones with correctly targeted events within the 5′ homology by Southern using probe a. (c) Identification of ES clones with correctly targeted events within the 3′ homology by Southern using probe b. (d) Identification of ES cell clones containing either the M2 allele or the M3 allele by PCR. The primers used in the PCR are shown in panel a as short arrows.

Generation of Recql5^−/− ES cells. (a) A gene targeting strategy to replace exon 4 with a PGKHprt (Hprt) cassette. This strategy can be used to generate a true Recql5 knockout allele, the M4 allele, in a single targeting experiment. Specifically, the targeting of the remaining wild-type Recql5 allele in heterozygous Recql5 knockout ES cells containing a M3 allele is shown. This resulted in the generation of homozygous Recql5 knockout (Recql5^−/−) ES cells. Note the opposite directions of transcription for the Hprt cassette and Recql5. X, XbaI. (b) Identification of ES clones in which both Recql5 alleles have been targeted. Clones in which the remaining wild-type Recql5 allele was correctly targeted within the 5′ homology were identified by Southern using probe a. (c) Confirmation of the complete deletion of exon 4 in clones identified in panel b by Southern with an exon 4-specific probe (probe c). (d) Results of an RT-PCR experiment showing the absence of the expected 595-bp product from the normal Recql5 mRNA and the presence of a 489-bp product from an aberrant transcript in a homozygous Recql5 knockout (M3/M4) ES cell clone. The primers used in the RT-PCRs are indicated in panel a as short arrows.

Growth characteristics and responses to gamma radiation in mouse ES cells. (a) Growth curves of wild-type and Recql5^−/− ES cells. (b) Sensitivity to gamma radiation. The results of clonogenic survival assay experiments. Error bars represent standard deviations.

Spontaneous crossovers in mouse ES cells. (a to c) Representative metaphase spreads from wild-type, Recql5^−/−, and Blm^−/− cells showing SCE. (d) The results of statistical analysis of SCE frequencies. Average number of SCE per chromosome in wild type, Recql5^−/−, and Blm^−/− cells were calculated and compared. Error bars indicate standard errors of the mean, and P values were calculated by one-way analysis of variance (ANOVA) with posttest. (e to f) Two examples of multiradial structures (indicated by arrows) observed in Recql5^−/− cells, indicative of interchromosomal exchanges.

Genetic rescue of the SCE phenotype in Recql5^−/− ES cells. (a) A schematic illustration of wild-type Recql5 and M3 alleles, showing the locations of primers (arrows) used in the RT-PCRs. (b) The frequencies of SCE in wild-type, Recql5^−/−, and Recql5^−/− Res cells. Error bars indicate standard errors of the mean and P values were calculated by one-way ANOVA with posttest. (c) The results of semiquantitative RT-PCR analyses. Semiquantitative RT-PCRs were performed with total RNA extracted from wild-type (W) and Recql5^−/− Res (R) ES cells. Primers used in the RT-PCRs are indicated in panel a as arrows above the wild-type Recql5 allele. The expression of the Gapdh gene was used as an internal control to compare the levels of Recql5 expression among samples. The cycle numbers of RT-PCRs were indicated above or below the images, showing results for Recql5 and Gapdh, respectively.

Spontaneous SCE in Recql5^−/− Blm^−/− ES cells. (a) A representative metaphase spread with a high number of SCE. (b) The result of statistical analysis on the frequencies of SCE. The numbers of SCE per chromosome in Recql5^−/−, Blm^−/−, and Recql5^−/− Blm^−/− ES cells were compared. Error bars indicate the standard errors of the mean, and P values were calculated by one-way ANOVA with posttest. (c) The distributions of cells in categories of different ranges of SCE.

Generation of Recql5 knockout mice. (a) A schematic illustration of portions of the wild-type Recql5 allele, the M2 conditional knockout allele, and the M3 knockout allele. Regions between exons 2 and 6 are shown. X, XbaI; S, SacI. (b) Identification of mice carrying the M2 allele (+/M2) by Southern analysis using probe a. (c) Identification of mice carrying the M3 allele (+/M3) by PCR. (d) Identification of homozygous Recql5 knockout (M3/M3) mice among the progeny of a cross between heterozygous knockout (+/M3) mice by Southern analysis by using probe d. The positions of the primers (arrows) used in PCRs are indicated in panel a.

The frequencies of spontaneous SCE in primary MEF cells. The numbers of SCE per chromosome in wild-type and Recql5^−/− MEF cells were compared. Error bars indicate the standard errors of the mean, and P values were calculated by t test.