Volume 1, Issue 5, Pages 697-705 (April 1998) Meiotic Prophase Arrest with Failure of Chromosome Synapsis in Mice Deficient for Dmc1, a Germline-Specific RecA Homolog  Douglas L. Pittman, John Cobb, Kerry J. Schimenti, Lawriston A. Wilson, Deborah M. Cooper, Ember Brignull, Mary Ann Handel, John C. Schimenti  Molecular Cell  Volume 1, Issue 5, Pages 697-705 (April 1998) DOI: 10.1016/S1097-2765(00)80069-6

Figure 1 Targeted Mutagenesis of Dmc1 (A) Gene targeting strategy. The restriction maps of the wild-type gene, the targeting construct, and the disrupted locus are shown. The asterisks correspond to exons encoding the RecA-like DNA binding motif GEFRTGKT. Lengths of certain targeting arms are shown in kilobases. The location of the 122 bp EcoRI-PvuII probe used to confirm correct targeting events by Southern blotting is indicated, as well as the size of the expected EcoRI restriction fragments. E = EcoRI; X = XbaI; P = PvuII; N/Jx refers to the NotI sites present at the junction between the phage vector polylinker and mouse DNA. The double-tailed arrow within the 5 kb deleted region corresponds to PCR primers (DP2 and DP18) used to identify targeted recombinants in (B). The small arrows under the wild-type and mutated locus structures are PCR primers (Dmc.3 and Dmc.4) used in the RT-PCR reaction in (C). The 5′-most primer begins at the “T” in the ATG start codon of Dmc1. (B) Detection of a targeted Dmc1 allele in ES cells. In the left panel is PCR amplification of DNA samples of the indicated genotypes using the primers DP2 and DP18. As shown, the 129 allele was deleted in the 129 × B6 ES cells, which is consistent with the use of targeting DNA that was derived from a 129/SvJ library (teRiele et al. 1992). In the right panel is a Southern hybridization of the probe shown in (A) with EcoRI digested genomic DNA. A correctly targeted allele resulted in a 5.3 kb fragment. (C) RT-PCR of testis RNA from Dmc1 knockout mice using primers Dmc.3 and Dmc.4. Lane 1, Dmc1+/+; lane 2, Dmc1−/−. Separate RT-PCR reactions of identical RNA samples were used for the Dmc1 and the control gene, Aop2 (see Experimental Procedures). Molecular Cell 1998 1, 697-705DOI: (10.1016/S1097-2765(00)80069-6)

Figure 2 Histology of Gonads of Dmc1-Deficient Mice Sections of testes and ovaries from animals of various ages are shown. (A)–(D) are sections through testes of 26-day-old littermates. Thin arrows indicate spermatocytes; the thick arrow indicates a round spermatid; the arrowhead indicates an elongated spermatid. (A) Dmc1+/−, 59.2× magnification. (B) Dmc1−/−, 78.1×. (C) Dmc1+/+, 225×. (D) Dmc1−/−, 226×. Note the mass of Sertoli cell cytoplasm in the Dmc1−/− tubules exposed by the absence of postmeiotic cells. (E)–(F) are sections through ovaries of 31-day-old littermates. The thin arrow indicates a primordial follicle, and the thick arrow a developing secondary follicle. (E) Dmc1+/+, 118.4× magnification. (F) Dmc1−/−, 118.4×. No oocytes or follicles are present. (G)–(H) are sections through embryonic ovaries of 17-day-pc siblings. Arrows indicate oocytes. The insets show representatives of the most advanced oocytes in each sample. (G) Dmc1+/+, 118.4× magnification, except for inset. The bar in the inset is 5 μM. The oocytes in the inset are in pachynema. (H) Dmc1−/−, 118.4×, inset as in (G). Molecular Cell 1998 1, 697-705DOI: (10.1016/S1097-2765(00)80069-6)

Figure 3 Immunofluorescence Analysis of Spermatocytes from Wild-Type and Dmc1 Knockout Mice Wild-type (A–C) and Dmc1−/− (D–F) spermatocytes were stained with anti-COR1, recognizing mouse SYCP3 (A and D), anti-SYN1, recognizing mouse SYCP1 (B and E), and anti-RAD51 (C and F). The cells were derived from littermates produced from a cross of Dmc1 heterozygotes. In (A), note normal patterns of staining with anti-SYCP3 for zygotene (Z), pachytene (P), and metaphase I (MI) spermatocytes. In (D) are three mutant zygotene-like spermatocytes showing characteristics of the early (E), mid (M), and late (L) zygotene stages. However, even where full axial elements are formed (L), no intimate pairing of axes is seen. These same cells are viewed with anti-SYCP1 staining in (B) and (E). In (B), immunoreactivity for SYCP1 is seen over all axes in wild-type pachytene spermatocytes (P) and over some axes (arrow) in zygotene spermatocytes (Z). However, in (E), no SYCP1-axes are seen in the mutant spermatocytes. In (C) is seen the normal pattern of fewer RAD51 foci (red) in pachytene spermatocytes colocalizing with the SC seen with anti-SYCP3 staining (green); note the dense foci on the X chromosome (X). In Dmc1−/− spermatocytes (F), numerous red RAD51 foci are seen, as in normal zygotene spermatocytes. Virtually all are associated with the axial elements, visualized with green anti-SYCP3 staining. Bars = 10 μm. Molecular Cell 1998 1, 697-705DOI: (10.1016/S1097-2765(00)80069-6)

Figure 4 Crossing over Frequency in Dmc1+/− Spermatocytes Male chimeras created with either Dmc1+/− ES cells or untargeted ES cells (“control”) were crossed to C57BL/6J females, and progeny that inherited ES cell-derived genomes were scored for recombination along chromosomes 10 and 18. The thick vertical line represents each chromosome, and the closed circle indicates the position of the centromere. Microsatellite loci that were typed in the crosses are indicated at the right of each chromosome, whereby the prefixes “D10Mit” and“ D18Mit” have been abbreviated with “M.” The numbers listed in the intervals between microsatellites are recombination percentages in those particular intervals. Control values are listed on the left, and Dmc1 chimeras on the right. The overall recombination frequency between the most distal and proximal markers on each chromosome are shown under the horizontal lines near the bottom. The results of chi2 analysis in a two-way contingency test are shown at the bottom. This reflects the probability that the overall recombination frequency in Dmc1+/− chimeras differed from untargeted control chimeras. The lengths of each chromosome as depicted do not reflect true physical or genetic size. Molecular Cell 1998 1, 697-705DOI: (10.1016/S1097-2765(00)80069-6)