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. Author manuscript; available in PMC: 2011 Dec 1.
Published in final edited form as: Genesis. 2010 Oct 30;48(12):717–722. doi: 10.1002/dvg.20675

Postnatal Male Germ Cell Expression of Cre Recombinase in Tex101-iCre Transgenic Mice

Zhenmin Lei 2,1, Jing Lin 2, Xian Li 2, Shengqiang Li 2, Huaxin Zhou 3, Yoshihiko Araki 4, Zi-Jian Lan 3,1
PMCID: PMC3005014  NIHMSID: NIHMS246800  PMID: 20853429

Abstract

We have generated a transgenic mouse line which expresses improved Cre recombinase (iCre) under the control of the testis-expressed gene 101 (Tex101) promoter. This transgenic mouse line was named Tex101-iCre. Using the floxed ROSA reporter mice, we found that robust Cre recombinase activity was detected in postnatal testes with weak or no activity in other tissues. Within the testis, Cre recombinase was active in spermatogenic cells as early as the prospermatogonia stage at day 1 after birth. In 30- and 60-day-old mice, positive Cre recombinase activity was detected not only in prospermatogonia but also in spermatogenic cells at later stages of spermatogenesis. There was little or no Cre activity in interstitial cells. Breeding wild-type females with homozygous floxed fibroblast growth factor receptor 2 (Fgfr2) males carrying the Tex101-iCre transgene did not produce any progeny with the floxed Fgfr2 allele. All of the progeny inherited a recombined Fgfr2 allele, indicating that complete deletion of the floxed Fgfr2 allele can be achieved in the male germline by Tex101-iCre mice. Furthermore, FGFR2 protein was not detected in spermatocytes and spermatids of adult Fgfr2fl/fl;Tex101-iCre mice. Taken together, our results suggest that the Tex101-iCre mouse line allows the inactivation of a floxed gene in spermatogenic cells in adult mice, which will facilitate the functional characterization of genes in normal spermatogenesis and male fertility.

Keywords: testis, iCre recombinase, prospermatogonia, spermatogenesis, promoter

Our goal is to characterize the gene regulatory network within spermatogenic cells (e.g., spermatocytes and round spermatids) during postnatal development. To achieve this goal, we want to adapt the Cre/loxP system (Sauer and Henderson, 1989; Gu et al., 1994) to uncover the physiological functions of spermatogenic cell-expressing genes, especially those genes (e.g., the germ cell nuclear factor gene) that are critical for embryonic development (Chung et al., 2001; Fuhrmann et al., 2001). One critical factor in the generation of spermatogenic cell-specific knockouts using the Cre/loxP strategy is the activity of Cre recombinase expressed in Cre transgenic mice. Several lines of Cre transgenic mice such as TNAP-Cre, Vasa-Cre, Stra8-iCre, TSPY-Cre, Pgk2-Cre, Sycp1Cre, c-kit-Cre, Enolase-Cre, Ngn3-Cre, Protamine-Cre, and SynCre (Lee et al., 2001; Rempe et al., 2006; Hammond and Matin, 2009) have been successfully generated. These mice can express Cre recombinase either in primordial male germ cells in embryos or in spermatogonia, spermatocytes, round spermatids, and/or elongating spermatids in postnatal testes. In our previous effort to characterize the function of germ cell nuclear receptor, we have used TNAP-Cre, Sycp1Cre, and Enolase-Cre mouse lines but failed to completely delete the floxed germ cell nuclear receptor gene in spermatocytes and round spermatids (Lan & Cooney, unpublished data). Incomplete deletion of floxed genes in male germ cells by TNAP-Cre and Sycp1Cre have also been observed in other laboratories (Rassoulzadegan et al., 2002; Kimura et al., 2003; Rasoulpour and Boekelheide, 2006). To date, successful use of the aforementioned Cre transgenic mice to completely and specifically delete floxed genes in spermatocytes and round spermatids has not yet been reported. This information prompted us to generate a Cre transgenic mouse line that can express Cre in spermatocytes and round spermatids at a high level. In this report, we describe the generation and characterization of a Cre transgenic mouse line driven by the promoter of testis-expressed gene 101 (Tex101) (Kurita et al., 2001; Takayama et al., 2005; Tsukamoto et al., 2007). A codon-improved Cre recombinase (iCre) gene (Shimshek et al., 2002), instead of prokaryotic Cre (Gu et al., 1994), was used to enhance the expression level of Cre recombinase and improve the recombination efficiency of loxP sites.

Tex101, a germ cell-specific gene, is expressed in prospermatogonia before the initiation of spermatogenesis (Takayama et al., 2005). During the first wave of spermatogenesis, Tex101 is highly expressed in spermatocytes and spermatids as well as spermatozoa, but not in spermatogonia, Sertoli cells, or interstitial cells (Takayama et al., 2005). The Tex101 promoter (−5779 to +19) fragment (Tsukamoto et al., 2007) was inserted into the promoter-less mini-iCre expression cassette, pGL3B-iCre (Lan et al., 2004), to generate the transgene vector, pGL3B-Tex101-iCre. Microinjection of the Tex101-iCre transgene (Fig. 1a), which contains the 5.8-kb Tex101 promoter, the 1.1-kb iCre coding sequence (Shimshek et al., 2002), and a SV40 late poly(A) signal, into the pronuclei of fertilized eggs (from the intercross of C57-B6/SJL mice) followed by transfer of the microinjected eggs into pseudo-pregnant mothers, produced one male (mouse #1) and five female (mouse #2 to #6) pups (Fig. 1b–c). Genomic PCR analysis showed that three mice (#1, #2, and #5) carried the Tex101-iCre transgene (Fig. 1b–c). After breeding with wild-type mice, these three mice successfully transmitted the transgene to their offspring.

FIG. 1. Generation of Tex101-iCre mice.

FIG. 1

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(a) Schematic representation of the Tex101-iCre transgene. The mouse Tex101 promoter fragment (−5779 to +19) was inserted upstream of an improved Cre (iCre) expression cassette which contains a consensus Kozak sequence, simian virus 40 (SV40) large T nuclear localization signal, the iCre coding sequences, and a SV40 poly(A) tail. (b) Genomic PCR using the iCre-F and iCre-R primers showing the presence of iCre in three mouse lines. (c) Allele-specific PCR using the Tex101-F and iCre-R primers showing the presence of the Tex101-iCre transgene in all three mouse lines.

R26R reporter mice (Soriano, 1999) were used to determine the tissue and cell specificity of Cre recombinase in three germline-transmitted Tex101-iCre mouse lines. Breeding of these three lines of Tex101-iCre mice with R26R mice produced R26R and Tex101-iCre;R26R mice. A variety of tissues (e.g. heart, kidney, liver, lung, spleen, adrenal gland, pancreas, thymus, testis, and ovary) were collected from 60-day-old R26R and Tex101-iCre;R26R mice, and subjected to β-galactosidase staining. Positive blue staining in tissues indicates that Cre recombinase is active. As expected, no β-galactosidase staining was observed in the testis and other tissues of R26R mice (Fig. 2a, data not shown). In Tex101-iCre;R26R mice generated from the Tex101-iCre mouse lines #1 and #5, positive blue staining was observed in all tested tissues (data not shown), indicating ectopic expression of Cre recombinase in these two mouse lines. However in Tex101-iCre;R26R mice generated from the Tex101-iCre mouse line #2, robust positive β-galactosidase staining was observed in the testis (Fig. 2b). Spotty blue staining was also observed on the surface of the kidney but not other tested tissues including the ovary (Fig. 2c, data not shown). Negative staining in the ovary was also observed in Tex101-iCre;R26R mice (derived from the Tex101-iCre mouse line #2) at 17.5 days post-coitum (dpc) (Fig. 2d). These results suggest that Tex101-iCre line #2 expresses Cre recombinase predominantly in the testis, not the ovary.

FIG. 2. Beta-galactosidase staining showing Cre recombinase activity in spermatogenic cells of Tex101-iCre;R26R mice.

FIG. 2

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(a) Whole-mount staining of a 60-day-old R26R testis. (b–k) Beta-galactosidase staining of gonads from Tex101-iCre;R26R mice. (b) Whole-mount staining of a 60-day-old testis. (c) Whole-mount staining of a 60-day-old ovary. (d) Negative staining in the ovary at 17.5 dpc. (e) Negative staining in the testis at 17.5 dpc. (f) Positive and spotty staining in prospermatogonia (black arrow) at postnatal day 1. (g) Positive staining in prospermatogonia (black arrow) at postnatal day 8. (h–i) Positive staining in spermatogenic cells, but not interstitial cells (Int, indicated by the star symbol) at postnatal day 30. Panel (i) is a higher magnification of the tubule (black arrowhead) in panel (h). In panel (i), the black arrow indicates prospermatogonia; red arrow, spermatogonia; red arrowhead, spermatocyte (Sp); R, round spermatids. (j–k) Positive staining in spermatogenic cells in the testis at postnatal day 60. Panel (k) is a higher magnification of panel (j). Black arrow, prospermatogonia.

To determine the ontogeny of the expression of Cre recombinase, we performed β-galactosidase staining of testes from Tex101-iCre;R26R mice (derived from the Tex101-iCre mouse line #2) and R26R littermates at 17.5 dpc and at day 1, 8, 30, and 60 after birth. As anticipated, there was no blue staining in testicular sections from R26R mice (data not shown). In Tex101-iCre;R26R mice at 17.5 dpc, there was no positive blue staining in the testis (Fig. 2e). However, spotty positive blue staining was observed in some prospermatogonia in Tex101-iCre;R26R mice at day 1 after birth (Fig. 2f). At postnatal day 8, positive blue staining in prospermatogonia was more evident in the testis (Fig. 2g). At postnatal day 30, robust blue staining was observed in all seminiferous tubules, but not in interstitial cells (Fig. 2h). Within the seminiferous tubules, positive staining was observed in prospermatogonia, spermatogonia, spermatocytes, and round spermatids (Fig. 2i). Similarly, robust blue staining was observed in all seminiferous tubules, more specifically in spermatogenic cells including some prospermatogonia, in 60-day-old Tex101-iCre;R26R testes (Fig. 2j–k). These results are at least partly consistent with endogenous Tex101 expression in the testis (Takayama et al., 2005). Our results suggest that Cre recombinase in the Tex101-iCre mouse line #2 is active in prospermatogonia as early as at postnatal day 1 and in spermatogenic cells at later stages of spermatogenesis. Cre recombinase activity displayed in Tex101-iCre mice is earlier than in PGK-Cre, c-Kit-Cre, Enolase-Cre, Sycp1Cre, and Protamine-Cre mice. Thus, Tex101-iCre transgenic mice can be used to delete genes of interest in spermatogenic cells as early as the prospermatogonia stage in postnatal testis.

Having determined that Cre recombinase in the Tex101-iCre mouse line #2 can efficiently induce excision of the artificial DNA fragment in the ROSA locus, we investigated whether Cre recombinase in Tex101-iCre mouse line #2 can delete a floxed endogenous gene fragment in male germ cells. The floxed Fgfr2 mice (Yu et al., 2003) were used to test the efficiency of Cre recombinase in Tex101-iCre mice. Breeding of Tex101-iCre females with Fgfr2fl/fl males produced Tex101-iCre;Fgfr2fl/+ mice (Fig. 3a). Female Tex101-iCre;Fgfr2fl/+ mice were then crossed with male Fgfr2fl/fl mice to generate Tex101-iCre;Fgfr2fl/fl males. To determine whether the floxed Fgfr2 allele was completely deleted by Tex101-iCre in the male germline, we performed genotyping analysis of the progeny from the breeding of Tex101-iCre;Fgfr2fl/fl males with Fgfr2+/+ females. If Cre recombinase is active in the spermatogenic cells, the floxed Fgfr2 allele (Fgfr2fl) will be converted to the recombined Fgfr2delta allele regardless of the presence or absence of Tex101-iCre transgene in the progeny. Indeed, no floxed Fgfr2 allele was detected in 40 pups generated from three Tex101-iCre;Fgfr2fl/fl male mice, and a recombined Fgfr2delta allele was observed in all progeny. A representative genotyping result is shown in Fig. 3b. These results show that Cre recombinase expressed in adult Tex101-iCre males is sufficient to completely delete the floxed Fgfr2 allele in the male germline.

FIG. 3. Deletion of the floxed Fgfr2 allele by Tex101-iCre in the male germline and spermatogenic cells.

FIG. 3

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(a) A breeding scheme for generating Tex101-iCre;Fgfr2fl/fl male mice. (b) A representative PCR genotyping result of a litter of pups produced from breeding a Tex101-iCre;Fgfr2fl/fl male with a Fgfr2+/+ female. Note the lack of the Fgfr2fl allele and the presence of the Fgfr2delta allele in all pups, indicating complete deletion of the floxed Fgfr2 allele in the male germline, regardless of the presence of the iCre transgene in the progeny. (c) Immunohistochemical studies showing the expression of the FGFR2 protein in adult Fgfr2fl/fl (left panel) and Tex101-iCre;Fgfr2fl/fl (right panel) testes. Note the loss of the FGFR2 protein in spermatocytes (arrows) and spermatids (arrowheads), but not in interstitial cells of Tex101-iCre;Fgfr2fl/fl testes.

It has been shown that FGFR2 is expressed in spermatocytes and spermatids as well as interstitial cells within adult rat testis (Cancilla and Risbridger, 1998). To validate the deletion of Fgfr2 in spermatogenic cells within mouse testis, we performed immunohistochemical studies to examine FGFR2 expression in adult Tex101-iCre;Fgfr2fl/fl testes. As expected, FGFR2 protein was detected in spermatocytes, spermatids, and interstitial cells of control Fgfrfl/fl mice (Fig. 3c, left panel). In Tex101-iCre;Fgfr2fl/fl testes (Fig. 3c, right panel), the FGFR2 protein was also detected in interstitial cells. However, FGFR2 expression in spermatocytes and spermatids was not observed in Tex101-iCre;Fgfr2fl/fl littermates (Fig. 3c, right panel). These results suggest the complete deletion of the floxed Fgfr2 in spermatogenic cells but not in interstitial cells by Tex101-iCre, resulting in complete loss of the FGFR2 protein in spermatocytes and spermatids. However, no obvious defects in spermatogenesis and male fertility were observed in Tex101-iCre;Fgfr2fl/fl male mice (data not shown), indicating that FGFR2 in spermatocytes and spermatids is dispensable for normal spermatogenesis and male fertility. Regardless, our results suggest that Tex101-iCre transgenic mice (Line #2) can be used for conditional deletion of genes in spermatogenic cells to uncover their functions in spermatogenesis and male fertility.

In conclusion, we generated and characterized a Tex101-iCre mouse line that can express Cre recombinase in spermatogenic cells as early as the prospermatogonia stage. More importantly, we found complete deletion of the floxed Fgfr2 allele in spermatogenic cells in the adult testis. We believe that these Tex101-iCre transgenic mice will be useful to determine the physiological functions of genes during spermatogenesis. In addition, this transgenic mouse line can be used for conditional expression of transgenes specifically in spermatogenic cells as early as the prospermatogonia stage.

MATERIALS AND METHODS

Animals

Healthy adult C57B6/SJL mice and the floxed ROSA, R26R (on the genetic background of 129S1/SvImJ) (Soriano, 1999), were purchased from The Jackson Laboratories (Bar Harbor, Maine). The floxed Fgfr2 mice (on a mixed background of C57BL/6J X 129X1/SvJ) (Yu et al., 2003) were generously provided by Dr. David Ornitz (University of Washington, St. Louis, MO). All mice were maintained on a 14-hour light:10-hour dark cycle, with free access to food and water, in the vivarium of the University of Louisville. All experiments in this study were approved in advance by the Animal Welfare Committee of the University of Louisville.

Generation of Tex101-iCre Transgenic Mice

The Tex101 promoter fragment (−5779 to +19) was isolated from the pGL-cPK plasmid (Tsukamoto et al., 2007) by restriction digestion using PvuII and KpnI enzymes followed by Klenow incubation (in the presence of dNTPs). Meanwhile, the promoter-less mini-iCre expression cassette, pGL3B-iCre (Lan et al., 2004), which contains a consensus Kozak sequence (Kozak, 1986), the nuclear localization signal peptide of a simian virus 40 (SV40) large T antigen, and coding sequences of the iCre gene (Shimshek et al., 2002), was digested with KpnI and then incubated with the Klenow fragment to remove the 5′ overhang. The Klenow-treated Tex101 promoter fragment (−5779 to +19) was cloned into the blunted KpnI site of pGL3B-iCre, using the Stratagene T4 DNA ligation kit to generate the Tex101-iCre transgene expression vector pGL3B-Tex101-iCre. The nucleotide sequence of this transgene vector was confirmed by sequence analysis.

A 7.13-kb Sac I/Sal I DNA fragment from pGL3B-Tex101-iCre, which contains the 5.8-kb Tex101 promoter, 1.1-kb iCre coding sequence, and a SV40 late poly(A) signal (Fig. 1a), was purified from an agarose gel and then microinjected into the pronuclei of fertilized eggs (C57-B6/SJLF1 X C57-B6/SJLF1 zygotes). The microinjected eggs were then transferred to pseudo-pregnant mothers to generate Tex101-iCre mice.

Mouse Genotyping

Mouse tail genomic DNA was extracted as described previously (Lan et al., 2002). Tex101-iCre mice were genotyped by PCR analysis using iCre-F and iCre-R primers (Lan et al., 2004) or Tex101-F (5′-gttcaaccaatgagacctcg-3′) and iCre-R primers. PCR conditions for genotyping the Fgfr2+, Fgfr2fl, Fgfr2delta, and R26R allele were reported previously (Yu et al., 2003; Lan et al., 2004; Poladia et al., 2006).

β-galactosidase Staining

Tex101-iCre female mice were crossed with R26R male mice to generate Tex101-iCre;R26R mice. Gonads at 17.5 dpc and tissues from 60-day-old mice were collected and then subjected to β-galactosidase staining according to the manufacturer’s protocol (Specialty Media, NJ). Whole-mount stained gonads at 17.5 dpc were fixed in formalin, sectioned, and counter-stained with 0.1% nuclear fast red, as described previously (Lan et al., 2004). Testicular cryosections from mice at 1, 8, 30, and 60 days after birth were prepared and subjected to β-galactosidase staining, as described previously (Lan et al., 2009). Whole-mount tissues and testicular sections were examined under a Zeiss Axioskop 2 microscope (Carl Zeiss Inc., Thornwood, NY).

Immunohistochemistry

Cryosections of testes from 3-month-old Fgfrfl/fl and Tex101-iCre; Fgfr2fl/fl littermates were prepared as described previously (Lan et al., 2009). Immunohistochemical staining of these cryosections was performed using a rabbit polyclonal anti-FGFR2 antibody (sc-122, 1:250 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) and the Rabbit Vectastain ABC kit (Vector Laboratories, Burlingame, CA) according to the manufacturer’s protocol.

Acknowledgments

We thank Drs. Rolf Sprengel and David Ornitz for providing the pBlue:iCre plasmid and floxed Fgfr2 mice, respectively. This study was supported by NIH grant #5R01 HD57501-01 (to Zhenmin Lei) and NCRR COBRE Grant#2P20-RR/DE17702 (to ZJL).

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