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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2007 Feb 13;104(8):2596–2601. doi: 10.1073/pnas.0609282104

Adenovirus-mediated gene delivery into mouse spermatogonial stem cells

Masanori Takehashi *, Mito Kanatsu-Shinohara *,†, Kimiko Inoue ‡, Narumi Ogonuki ‡, Hiromi Miki ‡, Shinya Toyokuni §, Atsuo Ogura ‡, Takashi Shinohara *,
PMCID: PMC1815228  PMID: 17299052

Abstract

Spermatogonial stem cells represent a self-renewing population of spermatogonia, and continuous division of these cells supports spermatogenesis throughout the life of adult male animals. Previous attempts to introduce adenovirus vectors into spermatogenic cells, including spermatogonial stem cells, have failed to yield evidence of infection, suggesting that male germ cells may be resistant to adenovirus infection. In this study we show the feasibility of transducing spermatogonial stem cells by adenovirus vectors. When testis cells from ROSA26 Cre reporter mice were incubated in vitro with a Cre-expressing adenovirus vector, Cre-mediated recombination occurred at an efficiency of 49–76%, and the infected spermatogonial stem cells could reinitiate spermatogenesis after transplantation into seminiferous tubules of infertile recipient testes. No evidence of germ-line integration of adenovirus vector could be found in offspring from infected stem cells that underwent Cre-mediated recombination, which suggests that the adenovirus vector infected the cells but did not stably integrate into the germ line. Nevertheless, these results suggest that adenovirus may inadvertently integrate into the patient's germ line and indicate that there is no barrier to adenovirus infection in spermatogonial stem cells.

Keywords: gene therapy, germ cell, spermatogenesis

Spermatogenesis depends on the continuous proliferation of spermatogonial stem cells. Although the number of spermatogonial stem cells is very small (comprising only 0.2–0.3% in the mouse testis) (1, 2), these cells undergo self-renewing division and produce differentiated cells while maintaining an undifferentiated state. Because spermatogonial stem cells transmit genetic information to the offspring, the introduction of genetic material into spermatogonial stem cells results in permanent modification of the germ line.

However, initial attempts to introduce genetic material into spermatogonial stem cells met with little success in part because of a lack of methods for transducing genetic material into stem cells. The first reported evidence of germ-line transduction used retrovirus vectors, which have relatively high infection efficiency and have been widely used in the transduction of stem cells in several self-renewing tissues (3). Spermatogonial stem cells were infected with retrovirus vectors in vitro and transplanted into the seminiferous tubules for offspring production. Transplanted stem cells colonized the empty seminiferous tubules of infertile recipient testes and reinitiated spermatogenesis, eventually leading to the production of transgenic animals (4, 5). Although these results opened up an opportunity for in vitro genetic manipulation of spermatogonial stem cells, they revealed a serious safety concern in human somatic gene therapy. Inadvertent infection of germ-line cells by gene therapy vectors could lead to vertical germ-line transmission of the virus and potential insertional mutagenesis.

Adenovirus is another important type of viral vector that is used in human gene therapy (6). Unlike retrovirus vectors, which can infect only dividing cells, adenovirus has relatively high transduction efficiency in target cells and infects both dividing and nondividing cells (6). Although adenovirus vectors can be prepared at higher titer than retrovirus vectors and infect a large range of host cells, including hematopoietic stem cells or embryonic stem cells (7, 8), many in vitro and in vivo attempts to transduce spermatogenic cells have not provided evidence of infection. In particular, the absence of a functional assay to directly measure stem cell activity has interfered with a conclusive determination of whether spermatogonial stem cells can be infected by adenoviruses. Although viruses that are injected intravenously or into nongonadal tissues can reach mouse or human testes, only Sertoli cells are preferentially infected by intratesticular injection, and there is no evidence of germ cell infection (9–14). Furthermore, there is no sign of infection after direct in vitro exposure of spermatogenic cells or mature sperm to adenovirus (15, 16). These studies suggest that male germ cells, including spermatogonial stem cells, cannot be infected by adenovirus vectors.

The purpose of this study is to examine the feasibility of adenovirus-mediated gene delivery to spermatogonial stem cells. We hypothesized that adenovirus infection did not occur in previous studies because of inefficient exposure of germ cells (or stem cells) to adenovirus and/or low sensitivity of the detection methods. To overcome these problems, we took advantage of the ROSA26 reporter mouse strain, which can sensitively monitor Cre-mediated deletion (17). By adding Cre recombinase into cells, it is possible to excise loxP-flanked DNA sequences in transfected cells (17). An adenovirus expressing the Cre recombinase gene was used to infect an enriched population of spermatogonial stem cells in vitro, and infected cells were transplanted into the seminiferous tubules of infertile animals. Recipient testes were analyzed for reporter gene expression, and offspring DNA was examined for virus gene integration.

Results

Infection of Spermatogonial Stem Cells in Vivo.

In preliminary experiments we examined whether it is possible to infect spermatogonial stem cells in vivo. An adenovirus vector that expresses LacZ gene (AxCANLacZ) was introduced into the seminiferous tubules of immature and mature wild-type mice. In contrast to i.v. delivery, microinjection into seminiferous tubules allows more efficient direct exposure of germ cells to adenovirus. In particular, immature testes should provide better accessibility to spermatogonial stem cells because of the absence of a blood–testis barrier and multiple layers of germ cells (18). Previous studies using immature testes have shown efficient in vivo retrovirus infection of spermatogonial stem cells (19). However, when we analyzed the injected testes 4–8 weeks after adenovirus injection we found only patchy infection in the Sertoli cells that received adenovirus at the immature stage (Fig. 1A). Although staining was also found in mature testes, this was similarly localized exclusively in the Sertoli cells, and we were not able to obtain clear evidence of germ cell infection (Fig. 1B). Testes transduced with adenovirus vector occasionally showed signs of inflammation, but spermatogenesis and expression of the LacZ gene were observed in all treated testes. In agreement with these results, when we microinjected EGFP-expressing adenovirus (AxCANEGFP) into mature testes, none of the 12 injected males sired transgenic progeny after mating with wild-type females. Although >100 offspring were analyzed for EGFP expression, we did not observe EGFP fluorescence (data not shown).

Fig. 1.

Fig. 1.

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Adenovirus-mediated gene transfer into male germ cells. (A and B) Histological appearance of testes injected with AxCANLacZ at 7 days (A) or 4 weeks (B) after birth. Whole mounts of testes were stained 1 month after virus injection. (C–H) In vitro infection of immature testis cells (C and D), GS cells (E and F), and mGS cells (G and H) by AxCANEGFP. The cells were exposed to adenovirus overnight at 2.5 × 105 pfu/ml (C and D) or 1.8 × 105 pfu/ml (E–H), and EGFP fluorescence was examined 6 h (D) or 1 day (F and H) after infection. (I) Flow-cytometric analysis of immature testis cells 2 days after transduction of AxCANEGFP at 2.5 × 105 pfu/ml. EpCAM-positive spermatogonia cells showed EGFP fluorescence. Black line, control Ig; red line, specific antibody. (J) Flow-cytometric analysis of GS cells 3 days after transduction of AxCANEGFP. The values are mean ± SEM (n = 3). (K) Increase in GS cell number after adenovirus infection. After overnight infection, GS cells were cultured for 6 days. The values are mean ± SEM (n = 4). Although no significant difference in cell number was found at 6.0 × 103 pfu/ml, GS cell growth was inhibited at higher virus concentrations (P < 0.05 by t test). (L) The intensity of EGFP signals decreased during a 17-day culture. The cells were infected at 2.5 × 105 pfu/ml and passaged twice during this period. (Scale bars: 50 μm for A and B and 100 μm for C–H.)

Infection of Spermatogonial Stem Cells in Vitro.

We next sought to examine whether adenovirus can infect spermatogonial stem cells in vitro. We dissociated immature testes and cultured them in the presence of AxCANEGFP. The immature testes contain enriched populations of spermatogonial stem cells because they lack differentiated cells (18). After infection, EGFP-expressing cells were found on the next day in culture (Fig. 1 C and D). To determine whether germ cells could be infected by the adenovirus, the infected cells were analyzed by flow cytometry 2 days after infection. The analysis revealed that 13.3% of the EGFP-positive cells expressed the spermatogonia marker EpCAM (Fig. 1I) (20).

To further examine whether the virus can infect spermatogonial stem cells, we exposed AxCANEGFP to cultured spermatogonial stem cells or germ-line stem cells (GS cells) (21). These cells can undergo self-renewing division in the presence of glial cell line-derived neurotrophic factor in vitro, but they reinitiate spermatogenesis after transplantation into seminiferous tubules. After overnight infection with AxCANEGFP, most of the GS cell colonies showed EGFP fluorescence (Fig. 1 E, F, and J). The EGFP expression continued after several passages. Within 2–3 weeks the fluorescence was barely detectable, which suggested that the vector did not integrate stably in the genome of the GS cells (Fig. 1L). Although we did not find a significant effect of virus on the growth and morphology of GS cells at low virus concentrations (6 × 103 pfu/ml), exposure to higher concentrations of adenovirus had a negative effect on GS cell growth, and only 13% of the cultured cells could be recovered a t 6 × 105 pfu/ml 6 days after infection (Fig. 1K). Adenovirus could also infect ES-like multipotent GS cells (mGS cells) (Fig. 1 G and H) (22). These results established that spermatogonia can be infected in vitro by adenovirus vector.

Spermatogenesis from Adenovirus-Infected Stem Cells.

To establish that spermatogonial stem cells are infected by adenovirus, it is necessary to test whether infected cells can initiate and maintain long-term spermatogenesis by spermatogonial transplantation, which is the only functional assay for spermatogonial stem cells (23). However, we assumed that detecting signs of infection might be difficult in germ cell colonies because adenovirus generally remains as an episomal element and may disappear as single stem cells undergo multiple rounds of division to mature into spermatozoa (24). In agreement with this possibility, the intensity of EGFP fluorescence in the AxCANEGFP-infected GS cells decreased during passages (Fig. 1L), and the injection of AxCANEGFP-infected testis cells into seminiferous tubules of infertile adult recipient animals did not yield clear evidence of germ cell transduction (data not shown).

To establish a more sensitive method for detection of viral gene transduction, we took advantage of the Cre recombinase system (Fig. 2) (25). In this experiment, testis cells used for infection were collected from ROSA26 Cre reporter (R26R) mice that were 5–10 days old. At this stage, spermatogonial stem cells are enriched in the testis because of the absence of differentiated cells. The testis cells were exposed overnight to Cre-expressing adenovirus (AxCANCre) in vitro (26), and the infected cells were collected by trypsin digestion on the next day for transplantation into the seminiferous tubules of histocompatible recipient mice that were 5–10 days old, allowing the efficient colonization of donor cells (18). Successful Cre-mediated recombination in infected cells would result in the deletion of DNA sequences that are flanked by loxP sequences. Because ROSA26 promoter is active during spermatogenesis, the infected cells start to express LacZ gene after Cre-mediated recombination, which can be readily detected by X-Gal staining (27).

Fig. 2.

Fig. 2.

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Diagram of the experimental procedure. Testis cells from donor R26R mice were dissociated by trypsin digestion and infected in vitro by AxCANCre adenovirus. Cre-mediated recombination removed the neo cassette, and LacZ gene expression was initiated under the ROSA26 ubiquitous promoter. The infected cells were transplanted into infertile recipient testes. At 20 weeks after transplantation, recipient testes were mechanically dissociated, and spermatogenic cells were microinjected into oocytes to produce offspring. DNA from the offspring was analyzed by Southern blotting and PCR for integration of adenovirus.

Four separate experiments were performed, and a total of 17 testes in 14 recipient animals were microinjected with the cultured cells. PCR and Southern blot analyses of the cultured cells revealed that 49–76% of the cells underwent Cre-mediated recombination after overnight incubation with Cre-expressing adenovirus (Fig. 3). This deletion occurred only when the cells were exposed to AxCANCre (Fig. 3B), and sequence analysis of the cultured cells confirmed that loxP regions were maintained after culture without the presence of AxCANCre (data not shown). Three months after transplantation, testes were recovered from some of the recipients and stained for LacZ activity. LacZ-expressing colonies were found in all recipient testes, indicating that Cre recombinase was successfully expressed and induced recombination in spermatogonial stem cells (Fig. 4A). Histological analysis of the recipient testes showed normal-appearing spermatogenesis, and mature spermatozoa could be found in the germ cell colony (Fig. 4B). LacZ-expressing round and elongated spermatids were also observed. Because spermatogonial stem cells are the only cell type that can establish complete spermatogenesis after transplantation, these results show that spermatogonial stem cells infected with adenovirus vector could induce normal spermatogenesis.

Fig. 3.

Fig. 3.

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Deletion of floxed sequence in ROSA26 locus. (A) Diagram of the experimental design to detect deletion of floxed sequence by PCR and Southern blot analyses. (B) PCR analysis of the AxCANCre-mediated deletion by in vitro infection of immature testis cells. Deletion was detected only when the cultured cells were exposed to AxCANCre. Deletion did not occur when AxCANEGFP was used for infection. (C) Deletion efficiency of ROSA26 locus. Genomic DNA of infected cells was digested with EcoRV and hybridized with a ROSA26-specific probe (see Materials and Methods). Levels of percentage deletion, estimated by the intensity of each band, are indicated below.

Fig. 4.

Fig. 4.

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LacZ expression after Cre recombination. (A) Macroscopic appearance of a recipient testis that received Cre-infected testis cells 3 months after transplantation. Blue tubules represent colonization of donor stem cells that were infected by AxCANCre adenovirus. (B) Histological appearance of a recipient testis. Note complete spermatogenesis. LacZ expression was found in all spermatogenic cells. (C) Macroscopic appearance of X-Gal-stained brain, kidney, testis, and liver of F1 offspring from AxCANCre-infected spermatogonial stem cells. Note ubiquitous LacZ expression. (D) Southern blot analysis of EcoRV-digested tail genomic DNA from mature F1 offspring hybridized with a ROSA26-specific probe. Two offspring showed Cre-mediated recombination. (E) PCR analysis of the deletion. The deletion of floxed sequence was confirmed by PCR detection method, as shown in Fig. 3A. (F) Pedigree of an F1 male demonstrating transmission of parental genotype for three generations. Solid symbols indicate LacZ expression in these progeny. (Scale bars: 1 mm for A and 20 μm for B.)

Lack of Adenovirus Integration in Offspring from Infected Stem Cells.

To determine whether the germ cells from the infected cells are fertile, we used in vitro microinsemination, a technique commonly used to produce offspring in animals and humans (28, 29). Testes or epididymides were collected from three different recipients 7 months after transplantation of adenovirus-infected donor cells. Seminiferous tubules or epididymis were dissected by fine forceps and mechanically dissociated by repeated pipetting. The recovered round spermatids and elongated spermatids and spermatozoa were microinjected into oocytes from C57BL/6 (B6) × DBA/2 F1 (BDF1) females. After 24 h of culture, 23 of 123 (19%) of the embryos developed to the two-cell stage, and they were transferred into the oviducts of pseudopregnant ICR females. Of the 85 embryos transferred, 46 (54%) implanted in the uteri and 21 offspring were born (Table 1). Overall, offspring were obtained from three of four recipient males that were used in microinsemination, and PCR and Southern blot analyses revealed that successful recombination occurred in all 14 offspring that carried ROSA floxed allele (Fig. 4 D and E). Nine offspring, six males and three females, grew up to be normal fertile adults. When we examined the expression of the LacZ gene, three of the nine offspring showed X-Gal staining in skin biopsy. The ROSA26 promoter is ubiquitously active (17), and LacZ expression was also found in many organs (brain, liver, kidney, and testis) of the offspring (Fig. 4C). Normal offspring were produced from both males and females, and the parental genotype was transmitted in a Mendelian manner to subsequent generations after natural mating (Fig. 4F).

Table 1.

In vitro microinsemination with spermatogenic cells recovered from recipient W mice

Type of cells injected No. of embryos transferred* No. of embryos implanted (%) No. of pups (%)
Round spermatid 19 10 (53) 0 (0)
Elongated spermatid 6 4 (67) 0 (0)
Testicular sperm 50 27 (54) 19 (38)
Epididymal sperm 10 5 (50) 2 (20)
Total 85 46 (54) 21 (25)
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Data are combined results from three recipient animals.

*Embryos were cultured for 24 h and transferred at the two-cell stage.

To determine whether the adenovirus DNA integrated into the genome of the offspring, DNA was collected from placentas or tails of the offspring and analyzed by PCR and Southern blotting, using the probe from adenovirus genome. Viral DNA was not detected in any of the 21 offspring using either method, indicating that the adenovirus vector did not integrate into the germ line (Fig. 5).

Fig. 5.

Fig. 5.

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Lack of adenovirus integration in the mature F1 offspring. (A) Southern blot analysis of F1 DNA samples hybridized with a probe specific for adenoviral sequences (see Materials and Methods). Controls represent viral DNA in amounts equivalent to 0.1, 1, and 10 copies of viral DNA per diploid genome. (B) PCR analysis of the F1 DNA samples. The adenovirus-specific 310-bp fragment was amplified from the F1 DNA samples. Controls containing 0.15 μg of normal mouse DNA were spiked with viral DNA representing 0.01, 0.1, and one copy of the viral genome.

Discussion

Previous attempts failed to demonstrate the infection of spermatogenic cells by adenovirus vectors. In our recent study we found that introduction of adenovirus into the seminiferous tubules resulted in transduction of Sertoli cells, but there was no evidence of infection in germ cells, which are more abundant in the testes (13). This occurred even though germ cells express adenovirus receptors (30, 31). Likewise, other in vivo and in vitro studies also reported that only somatic cells, but not germ cells, are infected (9–16). Nevertheless, the current study now demonstrates that spermatogonial stem cells are susceptible to adenovirus infection.

Several factors led to successful adenovirus infection in spermatogonial stem cells. First, we used a sensitive reporter mouse strain for virus infection (17). Previous studies have relied on marker gene expression from virus vector or DNA analysis. Although these methods successfully detected retrovirus infection, we assumed that they might fail to detect transient infections or small amounts of infective viruses. In contrast, deletion of floxed sequences in the host genome is irreversible and does not require the presence of adenovirus at the time of detection. Second, we used a chicken β-actin and cytomegalovirus enhancer promoter that has been used to express exogenous genes in spermatogenic cells (32, 33). It is known that germ-line cells suppress viral promoters, and the use of these in previous studies may be one reason that adenovirus infection was not detected (34). Third, we used a higher concentration of spermatogonial stem cells. Previous studies used adult testes, in which the concentration of stem cells is significantly low (0.2–0.3%) (1, 2). In contrast, we used a single-cell suspension prepared from immature testis or GS cells, which lack differentiated germ cells and are more enriched for spermatogonial stem cells, leading to highly efficient transduction.

An important aspect of adenovirus-mediated gene delivery is its high transduction efficiency. Because it has been considered that spermatogenic cells are resistant to adenovirus infection, our current results were unexpected. In the present study most of the GS cell colonies showed EGFP expression after overnight incubation with AxCANEGFP; in the most successful case, 79% of the cells underwent Cre-mediated deletion. This far exceeds the efficiency of other methods, in which 2–30% of spermatogonial stem cells can be transduced (4, 5, 19). In contrast, previous studies have reported that adenovirus cannot infect spermatogenic cells even when they are exposed to high concentrations of adenovirus (15, 16). This discrepancy suggests that spermatogonial stem cells may differ from more differentiated spermatogenic cells in their susceptibility to adenovirus infection. Given that adenovirus cannot infect mature sperm even when they are exposed to high concentrations of adenovirus (16), it is possible that male germ cells acquire resistance to viral infection as they mature. On the other hand, a potential drawback of the present approach is its toxicity. In the present study adenovirus also influenced the growth rate at high concentrations of adenovirus, which could interfere with the genetic manipulation of spermatogonial stem cells. Nonetheless, the infected cells were still able to differentiate normally after spermatogonial transplantation, indicating that the virus infection did not compromise stem cell function.

Our results have important implications for human gene therapy. Although no evidence of stable adenovirus integration was found in the present study, several lines of evidence have shown that rare integration can still occur after adenovirus infection (at a frequency of 10−1 to 10−5 per cell) in somatic cells and preimplantation embryos (35, 36), which raises the possibility that stable viral integration may also occur in germ-line cells. Given our results, it may be necessary to reevaluate the frequency of accidental virus insertion using experimental animals. In particular, the possibility of germ-line integration likely increases when the technique is applied to treat male infertility. We previously have rescued infertile male animals with a gene therapy approach, in which a germ cell growth factor is delivered into defective Sertoli cells with an adenovirus vector (13). The injected animals reinitiated spermatogenesis and sired offspring that did not show viral integration. This is currently the only method to rescue infertility because of Sertoli cell defects, and clinical application of this technique may rescue patients with severe hypospermatogenesis or few germ cells. Although the low frequency of stable integration and preferential infection of Sertoli cells in vivo suggest that adenovirus vectors may provide a relatively safe approach for treating Sertoli cell-based infertility, our current results indicate that caution is necessary when extrapolating this technique to clinical cases. Further studies are necessary to test whether such stable infection occurs in the male germ line.

One direct application of our results in basic research is using this in vitro infection system for analyzing gene functions in spermatogenesis. Although several Cre transgenic lines are available for studying spermatogenesis, very few are available for analysis of the spermatogonia stage, and it is often difficult to study gene functions in spermatogonial stem cells because they are identified only by their function to self-renew. In this sense, adenovirus-mediated Cre expression in spermatogonial stem cells may be useful. By transplanting transgenically marked cells, it is possible to visualize the pattern and kinetics of the repopulation process from single stem cells (27), which can reveal abnormal functions that are not evident in physiological conditions. The Cre infection may also be applied for selectable marker removal after gene targeting in GS cells (37) or for more sophisticated genetic modifications. For example, a transposon/transposase construct may be used as a cargo for adenovirus, and the ensuing transient expression of the cargo should generate an active transposase, which then inserts the transposon cargo permanently in the germ line. Thus, the unique mode of gene delivery by adenovirus vectors complements previously established genetic manipulation methods that achieve stable germ-line integration and will provide new opportunities for studies on male germ cell biology.

Materials and Methods

Recombinant Adenovirus.

The replication-defective adenovirus vectors AxCANLacZ and AxCANCre were obtained from RIKEN. AxCANEGFP was a generous gift from I. Saito (University of Tokyo, Tokyo, Japan). These vectors used the cytomegalovirus enhancer promoter, which can be expressed in spermatogonial stem cells (32, 33). The viruses were purified from 293 cells by using CsCl centrifugation. The titer of the virus was 2 × 108 pfu/ml, which was diluted before use.

Animals and Cell Culture.

For in vivo infection, purified adenovirus vector (1 × 106 pfu/ml) was microinjected into the seminiferous tubules of ICR mice that were 5–10 days and 5 weeks old (Japan SLC, Shizuoka, Japan). For in vitro infection, adenovirus was exposed to primary testis cells that were recovered from 6-day-old ICR mice or GS or mGS cells established from newborn DBA/2 mice (21, 22). All primary testis cells were maintained on mouse embryonic fibroblasts in Stempro34 medium (Invitrogen, Carlsbad, CA), as described (21). mGS cells were maintained in DMEM/15% FCS with leukemia inhibitory factor on gelatin-coated plates, and GS cells were cultured on laminin in Stempro34 medium, as described (38). In some experiments, we used testis cells from a R26R mouse that was kept in B6 background (The Jackson Laboratory, Bar Harbor, ME) (17). In infection of primary testis cells, 1 × 106 cells were plated in a 6-well plate (9.5 cm2), whereas 3 × 105 cells were plated in a 12-well plate (3.8 cm2) in infection of GS and mGS cells. The cells were incubated overnight with adenovirus at concentrations ranging from 6 × 103 to 6 × 105 pfu/ml. The in vitro deletion efficiency was estimated by using homozygous R26R mice, whereas heterozygous R26R mice were used to examine germ-line integration in the offspring. The cultured cells were transplanted into WBB6F1-W/Wv (W) mice that do not have endogenous spermatogenesis because of mutations in the c-kit gene (39).

Surgical Procedure.

Microinjection into the seminiferous tubules was performed via the efferent duct. Each injection filled 75–85% of the seminiferous tubules in each testis (40). Approximately 10 μl was introduced into the ICR testes, whereas only 2 or 4 μl could be introduced into W mice that were 5–10 days or 4 weeks old, respectively. When recipient mice were not histocompatible with the transplanted cells, they were treated with anti-CD4 antibody to induce tolerance to the donor cells (41). The Institutional Animal Care and Use Committee of Kyoto University approved all of the animal experimentation protocols.

Analysis of Transgene Expression.

In experiments using AxCANLacZ, the tissues were fixed in 4% paraformaldehyde for 2 h, and X-Gal staining was used to detect LacZ expression (27). The same procedure was used to detect LacZ expression in R26R mice after Cre recombination. In experiments using AxCANEGFP, cells were analyzed by a microscope equipped with UV fluorescence (21). For flow cytometry, the cultured cells were dissociated by trypsin, and single-cell suspensions were incubated with rat anti-mouse EpCAM antibody (G8.8; BD Biosciences, Franklin Lakes, NJ), which was detected by allophycocyanin-conjugated anti-rat IgG antibody (BD Biosciences), as described previously (42). The cells were analyzed by a FACSCalibur system (BD Biosciences).

Histological Analysis.

The testes were fixed with 10% neutral-buffered formalin and processed for paraffin sectioning. Two histological sections were made from each recipient testis with an interval of 12 μm between sections. All sections were stained with hematoxylin and eosin.

DNA Analysis.

Genomic DNA was isolated from cultured cells or tissue samples by phenol/chloroform extraction, followed by ethanol precipitation. The deletion of the floxed allele was estimated by PCR using the 5′-TTTCTGGGAGTTCTCTGCTGC-3′ and 5′-TCACGACGTTGTAAAACGACG-3′ primers.

To estimate the efficiency of Cre-mediated deletion, a 270-bp fragment in the ROSA26 promoter region was amplified by PCR using the 5′-CCTAAAGAAGAGGCTGTGCTTTGG-3′ and 5′-CGTCCGGTGGAGACTTTTC-3′ primers, which were used as a hybridization probe. Twenty micrograms of DNA was digested with restriction enzymes and separated on a 1.0% agarose gel. DNA transfer and hybridization were performed as described previously (33). To detect the adenovirus genome in offspring, a 15,043-bp ScaI-EcoRI fragment of pAxCAiLacZit cosmid vector was used as a hybridization probe (Nippon Gene, Toyama, Japan). The intensity of bands was quantified by NIH Image 1.63. To detect virus integration, a 310-bp region of adenovirus type 5 was amplified by PCR using specific primers, as described previously (13).

Microinsemination.

The seminiferous tubules of recipient mice were mechanically dissected, and spermatogenic cells were collected. Microinsemination was performed by intracytoplasmic injection, as described previously (28). Embryos that reached the two-cell stage after 24 h in culture were transferred to the oviducts of day-1 pseudopregnant ICR female mice. Fetuses that were retrieved on day 19.5 were raised by ICR foster mothers.

Acknowledgments

We are grateful to Ms. A. Wada for her technical assistance. We also thank Dr. M. Ikegawa for providing us with pAxCAiLacZit cosmid vector. Financial support for this research was provided by the Ministry of Education, Culture, Sports, Science, and Technology of Japan and by grants from CREST and the Human Science Foundation (Japan). This work was also supported in part by the Tokyo Biochemical Research Foundation, the Genome Network Project, and Special Coordination Funds for Promoting Science and Technology from the Ministry of Education, Culture, Sports, Science, and Technology.

Abbreviations

GS cell

germ-line stem cell

mGS cell

multipotent GS cell.

Footnotes

The authors declare no conflict of interest.

This article is a PNAS direct submission.

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