Targeted JAM-C deletion in germ cells by Spo11-controlled Cre recombinase

Abstract

Meiosis is a crucial process for the production of functional gametes. However, the biological significance of many genes expressed during the meiotic phase remains poorly understood, mainly because of the lethal phenotypes of the knockout mice. Functional analysis of such genes using the conditional knockout approach is hindered by the lack of suitable Cre transgenic lines. We describe here the generation of transgenic mice expressing Cre recombinase under the control of the meiotic Spo11 gene. Using LacZ-R26(loxP) and EYFP-R26(loxP) reporter mice, we show the specific expression and activity of Cre during meiosis in males and females. Spo11(Cre) mice were then crossed with floxed Nbs1 and JAM-C mice to produce conditional knockouts. A strong reduction of Nbs1 and JAM-C protein levels was found in the testis. Although Nbs1-deleted mice developed minor gonadal abnormalities, JAM-C-knockout mice showed a spermiogenetic arrest, as previously described for the null mice. These results provide strong evidence that Spo11(Cre) transgenic mice represent a powerful tool for deleting genes of interest specifically in meiotic and/or in postmeiotic germ cells.

Fig. 1.

Generation and evaluation of Spo11-IRES-Cre mice. (A) BAC targeting of the murine Spo11 locus after the stop codon of the gene. The DNA fragment containing the homology regions ARM1 and ARM2 for the DNA recombination and the IRES-Cre-Neomycin (Neo) sequences was obtained by ScaI enzymatic digestion from the modified PL459 plasmid. The restriction enzymes used to insert the homology regions into the original PL459 plasmid are indicated. Spo11 exons are shown as red boxes, and exons 12 and 13 are magnified. Frt sites flanking the Neo cassette are shown. In the IRES-Cre sequence the location of primers used for Cre analyses is shown. CM(R) represents the chloramphenicol resistance gene used as a probe to identify founder mice. (B) Representative RT-PCR analysis of testis, lung, liver, spleen and thymus mRNA isolated from a 2-month-old mouse of the D5 founder line (top panel) and ovaries mRNA from 14.5 d.p.c. embryo (bottom panel), showing tissue-specific Cre mRNA expression. Actin PCR was used as a loading control.

Fig. 2.

Spo11-Cre is expressed in meiotic germ cells. (A) Semiquantitative PCR of cDNA prepared from 3, 5, 7, 10 d.p.p. and adult testes (left and middle) and from 12.5, 13.5, 16.5 d.p.c. and 1 d.p.p. ovaries (right). Cre and Spo11 alpha and beta isoform mRNA (Bellani et al., 2010; Romanienko and Camerini-Otero, 2000) amplifications are shown. Actin PCR was used as a loading control. (B) β-gal-stained sections of seminiferous tubules of adult testis counterstained with nuclear Fast Red. Black arrows indicate β-gal-negative spermatogonia and somatic Sertoli cells; black and white arrowheads show β-gal-positive pachytene spermatocytes and spermatids, respectively. Asterisk indicates lepto-zygotene spermatocytes. (C) EYFP expression in testis from a 21 d.p.p. male. White and yellow arrowheads indicate pachytene spermatocytes and spermatids, respectively. White asterisk shows early meiotic spermatocytes and white arrows indicate spermatogonia or preleptotene cells. (D) β-gal-stained sections of adult ovaries counterstained with nuclear Fast Red. PF, primordial follicles; PrF, primary follicles; SF, secondary follicles. (E) EYFP expression in fetal ovaries from 14.5 d.p.c. mice. Arrowheads indicate meiotic oocytes and the asterisk, early meiotic preleptotene cells. The cell types were recognized by differential Hoechst 33342 staining of the nuclei. Scale bars: 100 μm in B and D; 10 μm in E.

Fig. 3.

Nbs1^Δ/− mice do not show the canonical meiotic phenotype of hypomorphic Nbs1 mice. (A) Hematoxylin and eosin stained sections of ovaries from 5-week-old females. Scale bar: 100 μm. (B) Identification of Cre-mediated recombination by semiquantitative PCR of genomic DNA obtained from tail and testis of 2-week-old Nbs1^loxP/− and Nbs1^Δ/− mice. Nbs1 genotype was determined by C133, W123 and Mut2 primers, as previously described (Zhu et al., 2001). Primer pairs LoxP-F and LoxP-R show the extent of Cre-mediated deletion of loxP sites, whereas NB5F and 148-R were used for detection of Cre-mediated band (Reina-San-Martin et al., 2005). (C) Identification of Cre-mediated recombination. For PCRs we used sequential tenfold dilutions of genomic DNA (indicated by wedges) obtained from tail and testis of 3-week-old mice. As determined by semiquantitative PCR, 90–94% of the loxP-flanked Nbs1 alleles were deleted from the genome in Nbs1^Δ/− testis cells (middle). NBS primers C133 and W123 in the NBS 59 region were used as control to normalize for the amount of input DNA (top). Primer pairs NB5F and 148-R detected the Cre-mediated recombination band (bottom). Data are representative of three independent experiments. (D) Western blot analysis of murine Nbs1 in whole-cell extracts prepared from total testis (left panel), sperm and thymocytes (right) of Nbs1^Δ/− mice relative to Nbs1^loxP/− normalized to tubulin. Numbers reflect the intensity of bands representing the amount of Nbs1 protein. No reduction of the Nbs1 protein was evident in thymocytes. Data shown are representative of two independent experiments.

Fig. 4.

JAM-C^Δ/− mice display the postmeiotic phenotype of JAM-C-null mice. (A) Hematoxylin and eosin stained sections of testis from 10-week-old littermates showing the depletion of elongated spermatids in JAM-C^Δ/− relative to control JAM-C^loxP/− mice and the consequent spermatogenetic arrest. Scale bar: 100 μm. (B) Higher magnification of sections in A of seminiferous tubules from JAM-C^Δ/− and JAM-C^loxP/− mice. The white arrowhead indicates elongated spermatids in control littermate and the black arrowhead shows degenerating spermatids in JAM-C^Δ/− mice. Asterisks indicate spermatozoa in control and JAM-C^Δ/− mice. Scale bar: 10 μm. (C) Identification of Cre-mediated recombination by semiquantitative PCR of genomic DNA obtained from tails and sperm of JAM-C^loxP/− and JAM-C^Δ/− mice. JAM-C F1 and JAM-C R1 primer pairs show the extent of Cre-mediated deletion of loxP sites, whereas JAM-C F1 and JAM-C R4 were used for detection of the null allele, used as control to normalize for the amount of input DNA. The ratio between the intensity of the JAM-C^loxP/− bands in sperm and tail was arbitrarily considered 1. Data shown are representative of three independent experiments. (D) Western blot analysis of JAM-C in whole-cell extracts prepared from lung, spermatocytes (Spcytes) and spermatids (Sptids) of JAM-C^−/−, JAM-C^Δ/− mice relative to JAM-C^loxP/−. Numbers represents the ratio of residual JAM-C protein in JAM-C^Δ/− germ cells with respect to JAM-C^loxP/− germ cells, using either actin or Erk2 as loading controls. (E) Immunofluorescence in freshly isolated round spermatids showing polarized F-actin (phalloidin, red), in most of the control JAM-C^loxP/− cells (arrowheads), but disorganized in JAM-C-deleted spermatids.