Biol Reprod
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

BOR - Papers in Press, published online ahead of print June 9, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.031294
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
71/4/1202    most recent
biolreprod.104.031294v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kanatsu-Shinohara, M.
Right arrow Articles by Shinohara, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kanatsu-Shinohara, M.
Right arrow Articles by Shinohara, T.
Agricola
Right arrow Articles by Kanatsu-Shinohara, M.
Right arrow Articles by Shinohara, T.
BIOLOGY OF REPRODUCTION 71, 1202–1207 (2004)
DOI: 10.1095/biolreprod.104.031294
© 2004 by the Society for the Study of Reproduction, Inc.

Reproductive Technology

Transgenic Mice Produced by Retroviral Transduction of Male Germ Line Stem Cells In Vivo1

Mito Kanatsu-Shinohara3, Shinya Toyokuni4, and Takashi Shinohara2,3

Horizontal Medical Research Organization3 Department of Pathology and Biology of Diseases,4 Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Spermatogonial stem cells are the only stem cells in the postnatal body that can transmit parental genetic information to the offspring, making them an attractive target cell population for animal transgenesis. Although transgenic mice and rats were recently produced by retrovirus transduction of these cells in vitro, with transplantation of the transduced cells into infertile recipients, the difficulty of restoring fertility and preparing recipients using spermatogonial transplantation limits practical application of the technique. In this article, we describe a novel approach for producing transgenic animals by transducing spermatogonial stem cells in vivo using a retrovirus vector. Microinjection of retrovirus into immature seminiferous tubules resulted in the direct transduction of spermatogonial stem cells in situ, and the animals produced transgenic offspring after mating with females. Transgenic mice were produced in C57BL/6, BALB/C, A, and C3H backgrounds, with an average efficiency of 2.8%. The transgene was transmitted stably and expressed in the next generation. The technique overcomes the drawback of the in vitro-transduction approach, and will be useful as a novel method for producing transgenic animals as well as providing a means for analyzing the self-renewal and differentiation processes of spermatogonial stem cells in vivo.

developmental biology, gametogenesis, Sertoli cells, spermatogenesis, testis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Germ cells have the unique ability to transmit parental genome information to offspring. Germline modification has attracted significant attention in the last two decades because it provides a strategy to manipulate genes in vivo, and applications range from basic biomedical research to production agriculture [1–8]. Current techniques to modify germline cells are based on oocytes or eggs from females. Developments in embryo culture and transfer technology have provided the groundwork for female germline modification. Although the technique is most widely used in mice, attempts to use female germ cells for other animal species have been limited due to their different reproductive behavior and difficulty in obtaining and manipulating eggs [9]. It is difficult to obtain transgenic animals in more than 1% of injected embryos [9]. Therefore, there is clearly a need to establish new protocols for germline modification that have a wider range of application.

While the female germ cells cease to proliferate before birth, spermatogonial stem cells, which produce all male germ cells, have the ability to self-renew [10, 11]. These cells continue to proliferate throughout life and support spermatogenesis. Unlike differentiated germ cells that have a limited life-span, stem cell-based transgenesis has a clear advantage in that transfected stem cells will produce enormous numbers of transgenic sperm continuously. A single rat stem cell with a transgene will produce ~2000 transgenic spermatozoa per epithelial cycle [12], so numerous transgenic animals can be produced from a founder male. Toward this end, several groups have succeeded in producing transgenic animals by transducing spermatogonial stem cells in vitro. Spermatogonial stem cells from mice and rats were transduced with retrovirus during short-term culture and were transplanted into infertile recipient animals to produce spermatogenesis [13, 14]. By mating with females, recipients produced transgenic animals with an efficiency comparable with female-based transgenic methods.

Although this technique suggests the possibility of male germline manipulation, it has several limitations. A major drawback of the in vitro-transduction approach is the low fertility rate of the recipient animals following spermatogonial transplantation [15]. One of the reasons is that the ablation of endogenous spermatogenesis, which is a prerequisite for efficient colonization of donor cells [16], often damages the environment of the recipient testes for donor-cell colonization [17–20]. Furthermore, the absence of optimal culture conditions for stem cells results in a significant decrease in stem-cell number [21, 22] and contributes to lowered fertility; only 10–20% of stem cells survive in vitro for 1 wk [21, 22]. Due to the rejection of allogeneic donor cells [23–25], the application of spermatogonial transplantation is still limited in most other animal species in which immunocompatible recipients are not readily available. For these reasons, the efficiency of fertility restoration after spermatogonial transplantation is limited and prevents the practical application of the technique for transgenesis.

A potentially competitive alternative for producing transgenic animals with spermatogonial stem cells is to introduce genes into stem cells in vivo, as this approach does not require the transplantation or culture of stem cells. However, attempts at such direct transduction of spermatogonial stem cells have met with little success [26–32]. In one study, a transgene was integrated into the germline using in vivo electroporation, but the expression did not last long, indicating that the transgene was introduced into differentiated germ cells [33]. In another study, the transgene was not integrated into the germline and was found to be dominantly expressed in Sertoli cells [34]. The difficulty in transfecting spermatogonial stem cells in vivo cannot be explained only by the low number of stem cells in the testis (2–3 stem cells per 104 testis cells) [11, 35] because it was impossible with a more efficient virus-based approach. Microinjection of various types of virus vectors into seminiferous tubules of adult testes also failed to transduce germline cells in vivo [36, 37], and no animal studies have shown germline transmission using this approach. It is now thought that stem cells are protected in a germline niche, which inhibits the access of the transgenes or virus particles to stem cells [28, 36].

We postulated that the use of immature testis might allow the direct transduction of spermatogonial stem cells because the seminiferous tubules of the immature testis lack tight junctions [12], which would allow virus particles easier access from the luminal side of the seminiferous tubules to stem cells in the niche. Retrovirus particles were microinjected into immature mouse testes, and the possibility of producing transgenic offspring from the transduced stem cells was examined.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Microinjection Procedure

C57BL/6 (B6), BALB/C, C3H, and A mice were purchased from Japan SLC (Shizuoka, Japan). Both immature pups (5–10 days old) and adult (4–6 wk old) mice were used for virus injection. In some experiments, the operated mice were mated with wild-type B6 females to produce transgenic offspring. For the testicular injections, approximately 2 µl of Dulbecco modified Eagle medium/10% fetal calf serum (DMEM/FCS) containing retrovirus particles were introduced into the seminiferous tubules of an immature testis, while 10 µl were introduced into the tubules of a mature mouse testis because the latter is larger. Microinjection involved efferent duct injection [38] and filled 75–85% of the tubules in each recipient testis. Adult mice were anesthetized using an Avertin injection (640 mg/kg). The pups were placed on ice to cause hypothermia-induced anesthesia [39]. In some experiments, only one testis was microinjected to avoid prolonged exposure to ice. The average survival rate of pup recipients was 82% after the operation. The pups were returned to their dams after the operation and used for mating after at least 6 wk.

The Institutional Animal Care and Use Committee of Kyoto University approved all of the animal experimentation protocols.

Preparation of Retrovirus

A replication-defective ecotropic Moloney leukemia retrovirus, Gen–pgkßgal, was used to infect spermatogonial stem cells [40]. This retrovirus expresses the Escherichia coli LacZ gene under the promoter of the phosphoglycerate kinase 1 (Pgk) gene; it was previously used to infect spermatogonial stem cells [13, 28]. Virus particles were stably produced from GP+E86 retrovirus packaging cells in DMEM/FCS [40]. The original virus titer was 3 x 105 colony-forming units/ml on NIH 3T3 cells. Virus-conditioned medium was collected from 48-h cultures of confluent cells, passed through a 0.45-µm filter to remove contaminating cell debris, and frozen at –80°C until use. The procedure for virus concentration has been described [41]. In brief, freeze-thawed virus stock was supplemented with 10 µg/ml polybrene previously (Sigma), and 200 ml of viral supernatant was centrifuged for 16 h at 6000 x g. The virus pellet was suspended in 1–2 ml of DMEM/FCS using a 25-gauge needle, and aliquoted in Eppendorf tubes. The virus supernatant was further centrifuged for an additional 16 h at 6000 x g and suspended in 20–30 µl. The virus supernatant was used immediately after collection. The final titer of the retrovirus concentrate was ~109 colony-forming units/ml. All procedures for retrovirus preparation were performed at 4°C.

Analysis of Testes

To visualize infected cells, testes that received a retrovirus injection were stained for LacZ expression with 5-bromo-4-chloro-3-indolyl ß-D-galactoside (X-gal) (Wako Pure Chemical Industries, Osaka, Japan), as described [42]. A cluster of germ cells was defined as a colony when it occupied more than 50% of the basal surface of the tubule and was at least 0.1 mm long [42]. The efficiency of colonization was evaluated by counting the total number of colonies under a stereomicroscope. All sections were stained with hematoxylin-eosin. Statistical analysis was performed using Student t-test.

Southern Blot

Genomic DNA was isolated from tail samples from each offspring by phenol/chloroform extraction, followed by ethanol precipitation. Ten micrograms of DNA were digested with BamHI, and separated on a 1.0% agarose gel. DNA transfer and hybridization were performed according to a conventional protocol. The BamHI-ClaI fragment of the LacZ cDNA (~2200 base pairs) was used as a hybridization probe.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Infection of Spermatogonial Stem Cells by Retrovirus Injection into Seminiferous Tubules

There are multiple layers of germ cells and tight junctions between the Sertoli cells in mature seminiferous tubules [12]. By contrast, the immature testis has a single layer of spermatogonia and lacks tight junctions (Fig. 1). Based on these structural differences, we postulated that retrovirus microinjected into the immature seminiferous tubules would have better accessibility to spermatogonia, including stem cells, than it would when injected into mature seminiferous tubules. To test this possibility, concentrated Gen–pgkßgal retrovirus was microinjected into the seminiferous tubules of immature and mature testes of B6 mice, and the transduction efficiency was compared. The mice were sacrificed and their testes were stained for LacZ activity at 2 mo after microinjection to examine them for the presence of infected cells. While none of the eight mature testes showed LacZ staining (data not shown), 3 of 6 immature testes showed clusters of LacZ-positive cells (Fig. 2A), indicating successful retrovirus infection. The pattern of LacZ staining was variable in these testes (Fig. 2, B–E). Some of the LacZ-expressing clusters were long blue stretches of seminiferous tubules, resembling spermatogenic colonies from transplanted spermatogonial stem cells [16]. In spermatogonial transplantation into ablated recipients, germ cell colonies generally have a dark-staining region in the center flanked by weakly stained region at both ends, resulting in a symmetric appearance [42]. It is considered that stem cells are increasing in their number by self-renewing division at both ends, while starting to differentiate in the center [42]. However, the colonies found in the retrovirus-transduced testes had asymmetric ends and often had dark staining regions in the center, which indicated that germ cell differentiation had occurred (Fig. 2, B and C). In addition, there were other clusters of LacZ-positive cells scattered widely in the seminiferous tubules (Fig. 2, D and E). Histological analysis revealed that spermatogenesis with LacZ staining, confirmed that spermatogonial stem cells could be transduced using this approach (Fig. 2F).



View larger version (119K):
[in this window]
[in a new window]
  		FIG. 1. Macroscopic and histological appearance of immature and mature testes. Pup and adult testes: macroscopic appearance of (A) pup and (B) adult testis. Histological appearance of (C) pup and (D) adult testis. The arrows indicate spermatogonia. Note the absence of differentiated germ cells and the different structure of the seminiferous tubules in the pup testis. Bar = 1 mm (A, B), 50 µm (C, D). Stain: hematoxylin and eosin



View larger version (89K):
[in this window]
[in a new window]
  		FIG. 2. Retroviral transduction of spermatogonial stem cells. Concentrated retrovirus expressing the LacZ gene was microinjected into the seminiferous tubules. Testes were stained with X-gal to visualize LacZ gene expression. Blue tubules represent colonies from transduced spermatogonial stem cells. A) Macroscopic appearance of a testis transduced using retrovirus. B–E) Proliferation patterns of infected cells. B) A typical colony in a transduced testis. The colony is asymmetric at the ends. C) A similar asymmetric colony in the same testis. D) A cluster of blue cells (arrow) in a different testis. Note the different staining pattern at the end of the colony (arrowhead) as compared with those in B or C. E) A cluster of blue cells in a testis, which is similar to D. G) Histological appearance of a section of seminiferous tubules from a transduced testis. Note the normal-appearing spermatogenesis. Elongated spermatids are observed. G) Macroscopic appearance of a testis from #2B. H) Testis from an F1 offspring derived from mating #2B with a wild-type female, indicating germline transmission and transgene expression. Bar = 1 mm (A, G, H); 100 µm (B–E), 20 µm (F). Stain: hematoxylin and eosin

Transgenic Mouse Production from Retrovirus-Infected Spermatogonial Stem Cells

We next examined whether the infected stem cells could produce transgenic animals. We microinjected retrovirus into the seminiferous tubules of 5- to 10-day-old B6, C3H, BALB/C, and A mice (founder mice). At least two separate experiments were performed for each strain. After 6 to 8 wk, the animals were caged with 2–3 wild-type female mice to produce offspring. Although some of the injected animals could not sire offspring due to postoperative adhesion associated with the operation [39], an average of 86% of the animals that received retrovirus became fertile (Table 1). The weight of the testis was comparable with that of normal, untreated mice. All offspring resulting from mating with wild-type females were examined for the presence of the transgene using Southern blotting with a LacZ gene-specific probe, and 40–132 offspring from each founder mouse were examined over 6–16 mo after the operation.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Efficiency of transgenic mouse production from different mouse strains

In total, Southern blot analysis indicated that 26% (8/ 31) of the fertile animals sired offspring with the transgene during the analysis period. Assuming that only one construct became incorporated per stem cell, the percentage of transgenic animals produced from the founder males ranged from 0.8% (1/127) to 6.6% (4/61), with an average of 2.8% (Table 2). Because only half of the parental chromosomes will be transmitted from the spermatogonial stem cells to the offspring, the result indicates that 5.6% of the spermatozoa were derived from transduced stem cells. Although the numbers of animals might be too few to draw conclusions about the genetic effect of the strains used to transduce stem cells, 2 of 4 (50%) C3H mice produced transgenic progeny. By contrast, only 1 of 9 (11%) BALB/ C mice produced progeny with the LacZ transgene. The first transgene appeared in the progeny from a BALB/C founder mouse as early as 50 days after retrovirus injection, with an average of 114 days. The transgene originated from spermatogonial stem cells that had been infected with the retrovirus because spermatozoa generated from differentiated germ cells will disappear by 35 days [10–12].


View this table:
[in this window]
[in a new window]
  		TABLE 2. Transgenic mice from spermatogonial stem cells infected in vivo.a

The founder animals were killed after 259–479 days to examine the degree of retrovirus infection. LacZ staining was found in the testis regardless of whether the animals produced transgenic offspring (Fig. 2G). However, the number of blue colonies was significantly greater for founder animals that produced transgenic offspring than for those with nontransgenic progeny (3.44 ± 2.22 vs. 0.03 ± 0.03 colonies per testis; mean ± SEM; P < 0.05). On the other hand, no significant relationship was found between the number of colonies and the percentage of transgenic progeny from a founder mouse. Interestingly, in two of the recipients that produced transgenic offspring (#3I and #3J), no blue colonies were found, suggesting that the stem cell with the transgene may have ceased proliferation or disappeared (Table 2). Alternatively, the construct may have undergone gene silencing in these testes. However, a histological analysis of testes from other founder mice showed complete spermatogenesis with normal-appearing organization in areas of the seminiferous tubules expressing the transgene (data not shown). Expression of the LacZ transgene in hemizygous F1 mice was observed in several organs in different animals (Fig. 2H).

Finally, we examined whether the transgene can be transmitted to the next generation. We used both male and female offspring from #2B founder mice, and each transgenic mouse was mated with a nontransgenic wild-type B6 mouse to generate F2 offspring. Six of eight offspring from the male transgenic mouse and one of three offspring from the female mouse showed the presence of the transgene in a Southern blot analysis (Fig. 3), confirming the stable transmission of the transgene. The expression of the LacZ transgene in F2 animals was similar to that in the F1 generation.



View larger version (101K):
[in this window]
[in a new window]
  		FIG. 3. Southern blot analysis of BamHI-digested tail genomic DNA from pups derived from #2B. A) DNA from an F1 offspring derived from wild-type females and #2B male mouse. Two (female #29 and male #37) of fourteen representative offspring contained the LacZ transgene. B) Transmission of the viral transgene to the F2 generation. Both #29 (F1 female) and #37 (F1 male) transmitted the transgene to the F2 generation


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we demonstrated that spermatogonial stem cells can be transduced in vivo and produce transgenic animals. Several factors contributed to the success of our experiments. First, we used immature animals. While stem cells in the adult testis are separated from the lumen of the seminiferous tubules by tight junctions between Sertoli cells, stem cells in the pup are directly accessible from the luminal side due to the absence of tight junctions [12]. An additional advantage of immature animals is that, where stem cells in the adult are slowly dividing cells, those in the pups are actively dividing [13, 28], which increases susceptibility to virus transduction. Second, the number of stem cells contributed to restoring fertility. In spermatogonial transplantation, extensive colonization of donor cells is a prerequisite for fertility restoration. It is estimated that at least 15% of stem cells are required to restore fertility after ablation treatment [43]. However, this is difficult to achieve because the number of stem cells decreases during short-time culture [21, 22] and only a limited number of cells can be reintroduced into the seminiferous tubules. Moreover, the ablation of endogenous germ cells for transplantation not only damages the germ cell environment for fertility restoration [17, 20, 44], but also exerts systemic toxicity, leading to the death of the recipient animals [18, 19]. Indeed, the ablation protocol is difficult to optimize due to strain or age differences in the sensitivity to ablation [14, 18, 19, 45, 46]. By contrast, our approach uses intact, wild-type animals. Because their testes have normal numbers of stem cells, they could sire offspring efficiently after virus injection. Third is the retrovirus concentration. The virus titer in our study was significantly higher than those used in the previous study [13, 28], which was clearly beneficial for increasing the number of infected stem cells.

One of the important advantages of this technique is that it does not depend on the genetic background of the animals. The success of the in vitro-transduction approach is influenced by the genetic background, due to problems associated with spermatogonial transplantation. In fact, transgenic offspring from mouse spermatogonial stem cells have only been obtained from genetically infertile mutant recipients [13] due to the difficulty in host preparation and rejection of donor cells. By contrast, we were able to produce transgenic offspring in four different strains and suggest a promising approach for solving this problem. Although the percentage of animals that produced transgenic offspring was lower than that obtained by using the in vitro approach (22% in our study vs. 33–38% in previous studies) [13, 14], this might be improved by using higher titer preparations and a particular type of retrovirus, such as lentivirus [14, 36]. Because lentivirus can infect both dividing and nondividing cells, including germ cells [14, 47, 48], it has a clear advantage over Moloney leukemia virus, which can only infect dividing cells. Therefore, our technique will overcome the difficulties associated with the in vitro-infection approach and increase the opportunity to manipulate spermatogonial stem cells.

An interesting observation that resulted from our study was that of the variable pattern of LacZ staining in the retrovirus-infected testes. While the colonies that developed in stem cell-depleted testis are generally uniform and have symmetric ends [42], those observed in our study showed variable patterns and had asymmetric ends. Because the LacZ staining probably originated from the transduction of single stem cells, our results suggest that differentiation of germ cells from stem cells occurs in a different manner to that of the donor stem cells in an ablated environment with no endogenous germ cells. Perhaps the different morphological patterns of the colonies reflect organization of spermatogenesis at the border of two different segments or stages. This is in agreement with a recent report that the pattern of spermatogenesis from transplanted stem cells is influenced by the presence of other germ cells in the seminiferous tubules [16]. In fact, the colony patterns in our study showed remarkable resemblance to the donor cell-derived colonies observed in wild-type recipient testis after spermatogonial transplantation. Such an interaction between germ cells was previously shown in several other studies [16, 49, 50] and is thought to be mediated by chalone, tissue-specific inhibitor of stem cell proliferation [51]. Specific marking of individual stem cells by retrovirus has proven to be a useful method for the studies of stem cell dynamics in other self-renewing tissues [52, 53], and further studies using retrovirus transduction techniques will be helpful in understanding the dynamics of spermatogonial stem cells.

A potentially important application of our results is that of the transgenesis of animals, for which conventional transgenic technology has proved to be impossible or inefficient [9]. While the recent establishment of a long-term culture system for spermatogonial stem cells in mice and bulls may improve the in vitro-transduction method and potentially lead to new developments in animal transgenesis [54–56], no such culture system is available for the majority of other species, and the problems associated with spermatogonial transplantation need to be resolved. In particular, the ablation of endogenous germ cells is even more difficult in large animal species due to their large body size and different testicular structure and endocrinological environment [18, 24, 57]. Moreover, although several procedures to enrich stem cells have been established in rodents and could be used to enhance the fertility-restoration rate [46, 58–60], spermatogonial stem cells in other animals are less well characterized, and methods to enrich stem cells are not available. Because the microinjection technique has already been established for many animal species [24–26, 57, 61, 62], the microinjection of virus vectors into immature animals can be readily extended to other species. Immature testes in domestic animals generally exert less resistance to the flow of an injection into seminiferous tubules, which would facilitate the application of our technique [24–26, 57, 61, 62]. Therefore, the in vivo transduction of spermatogonial stem cells now provides a novel strategy for manipulating spermatogonial stem cells and stimulates new efforts geared toward understanding and using this valuable population of cells.


   ACKNOWLEDGMENTS
 
We thank Dr. P. Soriano for the retroviral producer cell line. We are grateful to Ms. Y. Doi, N. Tomikawa, and S. Hashino for their technical assistance.


   FOOTNOTES
 
1 Support for this research was provided by the Ministry of Education, Culture, Sports, Science and Technology of Japan. Back

2 Correspondence: Takashi Shinohara, Horizontal Medical Research Organization, Graduate School of Medicine, Kyoto University Yoshida-Konoe, Sakyo-ku, Kyoto 606-8501, Japan. FAX: 81 75 753 9306; takashi{at}mfour.med.kyoto-u.ac.jp Back

Received: 26 April 2004.

First decision: 19 May 2004.

Accepted: 3 June 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Palmiter RD, Brinster RL. Germ-line transformation of mouse. Ann Rev Genet 1986 20:465-499[CrossRef][Medline]
  2. Doetschman T, Gregg RG, Maeda N, Hooper ML, Melton DW, Thompson S, Smithies O. Targeted correction of a mutant HPRT gene in mouse embryonic stem cells. Nature 1987 330:576-578[CrossRef][Medline]
  3. Thomas KR, Capecchi MR. Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell 1987 51:503-512[CrossRef][Medline]
  4. Robertson E, Bradley A, Kuehn M, Evans M. Germ-line transmission of genes introduced into cultured pluripotential cells by retroviral vector. Nature 1986 323:445-448[CrossRef][Medline]
  5. Jaenisch R. Germ line integration and Mendelian transmission of the exogenous Moloney leukemia virus. Proc Natl Acad Sci U S A 1976 73:1260-1264[Abstract/Free Full Text]
  6. Perry AC, Wakayama T, Kishikawa H, Kasai T, Okabe M, Toyoda Y, Yanagimachi R. Mammalian transgenesis by intracytoplasmic sperm injection. Science 1999 284:1180-1183[Abstract/Free Full Text]
  7. Chan AWS, Homan EJ, Ballou LU, Burns JC, Bremel RD. Transgenic cattle produced by reverse-transcribed gene transfer in oocytes. Proc Natl Acad Sci U S A 1998 95:14028-14033[Abstract/Free Full Text]
  8. Schnieke AE, Kind AJ, Ritchie WA, Mycock K, Scott AR, Ritchie M, Wilmut I, Colman A, Campbell KH. Human factor IX transgenic sheep produced by transfer of nuclei from transfected fetal fibroblasts. Science 1997 278:2130-2133[Abstract/Free Full Text]
  9. Pinkert CA. Genetic engineering of farm animals. In: Hafez ESE, Hafez B (eds.), Reproduction in Farm Animals. Philadelphia, PA: Lippincott Williams and Wilkins; 2000:1–40
  10. de Rooij DG, Russell LD. All you wanted to know about spermatogonia but were afraid to ask. J Androl 2000 21:776-798[Medline]
  11. Meistrich ML, van Beek MEAB. Spermatogonial stem cells. In: Desjardins C, Ewing LL (eds.), Cell and Molecular Biology of the Testis. New York: Oxford University Press; 1993:266–295
  12. Russell LD, Ettlin RA, Sinha Hikim AP, Clegg ED. Mammalian spermatogenesis. In: Russell LD, Ettlin RA, Sinha Hikim AP, Clegg ED (eds.), Histological and Histopathological Evaluation of the Testis. Clearwater, FL: Cache River Press; 1990:1–40
  13. Nagano M, Brinster CJ, Orwig KE, Ryu B-Y, Avarbock MR, Brinster RL. Transgenic mouse produced by retroviral transduction of male germ-line stem cells. Proc Natl Acad Sci U S A 2001 98:13090-13095[Abstract/Free Full Text]
  14. Kent Hamra F, Gatlin J, Chapman KM, GrellhesI DM, Victor Garcia J, Hammer RE, Garbers DL. Production of transgenic rats by lentiviral transduction of male germ-line stem cells. Proc Natl Acad Sci U S A 2002 99:14931-14936[Abstract/Free Full Text]
  15. Brinster RL, Zimmermann JW. Spermatogenesis following male germ-cell transplantation. Proc Natl Acad Sci U S A 1994 91:11298-11302[Abstract/Free Full Text]
  16. Shinohara T, Orwig KE, Avarbock MR, Brinster RL. Germ line stem cell competition in postnatal mouse testes. Biol Reprod 2003 66:1491-1497
  17. Ogawa T, Dobrinski I, Brinster RL. Recipient preparation is critical for spermatogonial transplantation in the rat. Tissue Cell 1999 31:461-472[CrossRef][Medline]
  18. Brinster CJ, Ryu B-Y, Avarbock MR, Karagenc L, Brinster RL. Restoration of fertility by germ cell transplantation requires effective recipient preparation. Biol Reprod 2003 69:412-420[Abstract/Free Full Text]
  19. Ryu B-Y, Orwig KE, Avarbock MR, Brinster RL. Stem cell and niche development in the postnatal rat testis. Dev Biol 2003 263:253-263[CrossRef][Medline]
  20. Kanatsu-Shinohara M, Ogonuki N, Inoue K, Ogura A, Toyokuni S, Shinohara T. Restoration of fertility in infertile mice by transplantation of cryopreserved male germline stem cells. Hum Reprod 2003 18:2660-2667[Abstract/Free Full Text]
  21. Orwig KE, Avarbock MR, Brinster RL. Retrovirus-mediated modification of male germline stem cells in rats. Biol Reprod 2002 67:874-879[Abstract/Free Full Text]
  22. Nagano M, Ryu B-Y, Brinster CJ, Avarbock MR, Brinster RL. Maintenance of mouse male germ line stem cells in vitro. Biol Reprod 2003 68:2207-2214[Abstract/Free Full Text]
  23. Kanatsu-Shinohara M, Ogonuki N, Inoue K, Ogura A, Toyokuni S, Honjo T, Shinohara T. Allogeneic offspring produced by male germ line stem cell transplantation into infertile mouse testis. Biol Reprod 2003 68:167-173[Abstract/Free Full Text]
  24. Izadyar F, den Ouden K, Stout TAE, Stout J, Coret J, Lankveld DPK, Spoormakers TJP, Colenbrander B, Oldenbroek JK, van der Ploeg KD, Woelders H, Kal HB, de Rooij DG. Autologous and homologous transplantation of bovine spermatogonial stem cells. Reproduction 2003 126:765-774[Abstract]
  25. Joerg H, Janett F, Schlatt S, Mueller S, Graphodatskaya D, Suwattana D, Asai M, Stranzinger G. Germ cell transplantation in an azoospermic Kleinfelter bull. Biol Reprod 2003 69:1940-1944[Abstract/Free Full Text]
  26. Kim J-H, Jung-Ha H-S, Lee H-T, Chung K-S. Development of a positive method for male stem cell-mediated gene transfer in mouse and pig. Mol Reprod Dev 1997 233:45-49[CrossRef]
  27. Panthier JJ, Gounon P, Condamine H, Jacob F. Pattern of expression of ecotropic murine leukemia virus in gonads of inoculated SWR/J mice. J Virol 1989 63:2134-2142[Abstract/Free Full Text]
  28. Nagano M, Shinohara T, Avarbock MR, Brinster RL. Retrovirus-mediated gene delivery into male germline stem cells. FEBS Lett 2000 475:7-10[CrossRef][Medline]
  29. Huang Z, Tamura M, Sakurai T, Chuma S, Saito T, Nakatsuji N. In vivo transfection of testicular germ cells and transgenesis by using the mitochondrially localized jellyfish fluorescent protein gene. FEBS Lett 2000 487:248-251[CrossRef][Medline]
  30. Porada CD, Tran N, Eglitis M, Moen RC, Troutman L, Flake AW, Zhao Y, Anderson WF, Zanjani ED. In utero gene therapy: transfer and long-term expression of the bacterial neo(r) gene in sheep after direct injection of retroviral vectors into preimmune fetuses. Hum Gene Ther 1998 9:1571-1585[Medline]
  31. Soriano P, Gridley T, Jaenisch R. Retrovirus and insertional mutagenesis in mice: proviral integration at the Mov 34 locus leads to early embryonic death. Genes Dev 1987 1:366-375[Abstract/Free Full Text]
  32. Muramatsu T, Shibata O, Ryoki S, Ohmori Y, Okumura J-I. Foreign gene expression in the mouse testis by localized in vivo gene transfer. Biochem Biophys Res Commun 1997 233:45-49[CrossRef][Medline]
  33. Yamazaki Y, Fujimoto H, Ando H, Ohyama T, Hirota Y, Noce T. In vivo gene transfer to mouse spermatogenic cells by deoxyribonucleic acid injection into seminiferous tubules and subsequent electroporation. Biol Reprod 1998 59:1439-1444[Abstract/Free Full Text]
  34. Yomogida K, Yagura Y, Nishimune Y. Electroporated transgene-rescued spermatogenesis in infertile mutant mice with a Sertoli cell defect. Biol Reprod 2002 67:712-717[Abstract/Free Full Text]
  35. Tegelenbosch RAJ, de Rooij DG. A quantitative study of spermatogonial multiplication and stem cell renewal in the C3H/101 F1 hybrid mouse. Mutation Res 1993 290:193-200
  36. Ikawa M, Tergaonkar V, Ogura A, Ogonuki N, Inoue K, Verma IM. Restoration of spermatogenesis by lentiviral gene transfer: offspring from infertile mice. Proc Natl Acad Sci U S A 2002 99:7524-7529[Abstract/Free Full Text]
  37. Kanatsu-Shinohara M, Ogura A, Ikegawa M, Inoue K, Ogonuki N, Tashiro K, Toyokuni S, Honjo T, Shinohara T. Adenovirus-mediated gene delivery and in vitro microinsemination produce offspring from infertile male mice. Proc Natl Acad Sci U S A 2002 99:1383-1388[Abstract/Free Full Text]
  38. Ogawa T, Aréchaga JM, Avarbock MR, Brinster RL. Transplantation of testis germinal cells into mouse seminiferous tubules. Int J Dev Biol 1997 41:111-122[Medline]
  39. Shinohara T, Orwig KE, Avarbock MR, Brinster RL. Remodeling of the postnatal mouse testis is accompanied by dramatic changes in stem cell number and niche accessibility. Proc Natl Acad Sci U S A 2001 98:6186-6191[Abstract/Free Full Text]
  40. Soriano P, Friedrich G, Lawinger P. Promoter interactions in retrovirus vectors introduced into fibroblasts and embryonic stem cells. J Virol 1991 65:2314-2319[Abstract/Free Full Text]
  41. Bowles NE, Eisensmith RC, Mohuiddin R, Pyron M, Woo SL. A simple and efficient method for the concentration and purification of recombinant retrovirus for increased hepatocyte transduction in vivo. Hum Gene Ther 1996 7:1735-1742[Medline]
  42. Nagano M, Avarbock MR, Brinster RL. Pattern and kinetics of mouse donor spermatogonial stem cell colonization in recipient testes. Biol Reprod 1999 60:1429-1436[Abstract/Free Full Text]
  43. Meistrich ML, Hunter NR, Suzuki N, Trostle PK, Withers HR. Gradual regeneration of mouse testicular stem cells after exposure to ionizing radiation. Radiat Res 1978 74:349-362[Medline]
  44. Meistrich ML. Hormonal stimulation of the recovery of spermatogenesis following chemo- and radiotherapy. APMIS 1998 106:37-45[Medline]
  45. Brinster RL, Avarbock MR. Germline transmission of donor haplotype following spermatogonial transplantation. Proc Natl Acad Sci U S A 1994 91:11303-11307[Abstract/Free Full Text]
  46. Kubota H, Avarbock MR, Brinster RL. Spermatogonial stem cells share some, but not all, phenotypic and functional characteristics with other stem cells. Proc Natl Acad Sci U S A 2003 100:6487-6492[Abstract/Free Full Text]
  47. Kurita K, Burgess SM, Sakai N. Transgenic zebrafish produced by retroviral infection of in vitro-cultured sperm. Proc Natl Acad Sci U S A 2004 101:1263-1267[Abstract/Free Full Text]
  48. Nagano M, Watson DJ, Ryu B-Y, Wolfe JH, Brinster RL. Lentiviral vector transduction of male germ line stem cells in mice. FEBS Lett 2002 524:111-115[CrossRef][Medline]
  49. Clermont Y, Mauger A. Existence of a spermatogonial chalone in the rat testis. Cell Tissue Kinet 1974 7:165-172[Medline]
  50. de Rooij DG. Effect of testicular extracts on proliferation of spermatogonia in the mouse. Virchows Arch B Cell Pathol Include Mol Pathol 1980 33:67-80
  51. Bullough WS, Laurence EB. The control of epidermal mitotic activity in the mouse. Proc R Soc Lond B Biol Sci 1960 151:517-536[Medline]
  52. Jordan CT, Lemischka IR. Clonal and systemic analysis of long-term hematopoiesis in the mouse. Genes Dev 1990 4:220-232[Abstract/Free Full Text]
  53. Mackenzie IC. Retroviral transduction of murine epidermal stem cells demonstrates clonal units of epidermal structure. J Invest Dermatol 1997 109:377-383[CrossRef][Medline]
  54. Izadyar F, den Ouden K, Creemers LB, Posthuma G, Parvinen M, de Rooij DG. Proliferation and differentiation of bovine type A spermatogonia during long-term culture. Biol Reprod 2003 68:272-281[Abstract/Free Full Text]
  55. Kanatsu-Shinohara M, Ogonuki N, Inoue K, Miki H, Ogura A, Toyokuni S, Shinohara T. Long-term proliferation in culture and germline transmission of mouse male germline stem cells. Biol Reprod 2003 69:612-616[Abstract/Free Full Text]
  56. Jeon D, McLean DJ, Griswold MD. Long-term culture and transplantation of murine testicular germ cells. J Androl 2003 24:661-669[Abstract/Free Full Text]
  57. Schlatt S, Foppiani L, Rolf C, Weinbauer GF, Nieschlag E. Germ cell transplantation into X-irradiated monkey testes. Hum Reprod 2002 17:55-62[Abstract/Free Full Text]
  58. Shinohara T, Orwig KE, Avarbock MR, Brinster RL. Spermatogonial stem cell enrichment by multiparameter selection of mouse testis cells. Proc Natl Acad Sci U S A 2000 97:8346-8351[Abstract/Free Full Text]
  59. Giuili G, Tomljenovic A, Labrecque N, Oulad-Abdelghani M, Rassoulzadegan M, Cuzin F. Murine spermatogonial stem cells: targeted transgene expression and purification in an active state. EMBO Rep 2002 3:753-759[CrossRef][Medline]
  60. Kanatsu-Shinohara M, Toyokuni S, Shinohara T. CD9 is a surface marker on mouse and rat male germline stem cells. Biol Reprod 2004 70:70-75[Abstract/Free Full Text]
  61. Schlatt S, Rosiepen G, Weinbauer G, Rolf C, Brook PF, Nieschlag E. Germ cell transfer into rat, bovine, monkey and human testes. Hum Reprod 1999 14:144-150[Abstract/Free Full Text]
  62. Honaramooz A, Behboodi E, Megee SO, Overton SA, Galantino-Homer H, Echelard Y, Dobrinski I. Fertility and germline transmission of donor haplotype following germ cell transplantation in immunocompetent goats. Biol Reprod 2003 69:1260-1265[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Mol Hum ReprodHome page
A. J. Childs, P. T.K. Saunders, and R. A. Anderson
Modelling germ cell development in vitro
Mol. Hum. Reprod., September 1, 2008; 14(9): 501 - 511.
[Abstract] [Full Text] [PDF]

Home page
 ReproductionHome page
S. Goel, M. Fujihara, N. Minami, M. Yamada, and H. Imai
Expression of NANOG, but not POU5F1, points to the stem cell potential of primitive germ cells in neonatal pig testis
Reproduction, June 1, 2008; 135(6): 785 - 795.
[Abstract] [Full Text] [PDF]

Home page
 ReproductionHome page
J. Kalina, F. Senigl, A. Micakova, J. Mucksova, J. Blazkova, H. Yan, M. Poplstein, J. Hejnar, and P. Trefil
Retrovirus-mediated in vitro gene transfer into chicken male germ line cells
Reproduction, September 1, 2007; 134(3): 445 - 453.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
S. Goel, M. Sugimoto, N. Minami, M. Yamada, S. Kume, and H. Imai
Identification, Isolation, and In Vitro Culture of Porcine Gonocytes
Biol Reprod, July 1, 2007; 77(1): 127 - 137.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
M. Takehashi, M. Kanatsu-Shinohara, K. Inoue, N. Ogonuki, H. Miki, S. Toyokuni, A. Ogura, and T. Shinohara
Adenovirus-mediated gene delivery into mouse spermatogonial stem cells
PNAS, February 20, 2007; 104(8): 2596 - 2601.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
M. Kanatsu-Shinohara, K. Inoue, H. Miki, N. Ogonuki, M. Takehashi, T. Morimoto, A. Ogura, and T. Shinohara
Clonal Origin of Germ Cell Colonies after Spermatogonial Transplantation in Mice
Biol Reprod, July 1, 2006; 75(1): 68 - 74.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
M. Kanatsu-Shinohara, M. Ikawa, M. Takehashi, N. Ogonuki, H. Miki, K. Inoue, Y. Kazuki, J. Lee, S. Toyokuni, M. Oshimura, et al.
From the Cover: Production of knockout mice by random or targeted mutagenesis in spermatogonial stem cells
PNAS, May 23, 2006; 103(21): 8018 - 8023.
[Abstract] [Full Text] [PDF]

Home page
 Hum ReprodHome page
M. Kanatsu-Shinohara, H. Miki, K. Inoue, N. Ogonuki, S. Toyokuni, A. Ogura, and T. Shinohara
Germline niche transplantation restores fertility in infertile mice
Hum. Reprod., September 1, 2005; 20(9): 2376 - 2382.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
71/4/1202    most recent
biolreprod.104.031294v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kanatsu-Shinohara, M.
Right arrow Articles by Shinohara, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kanatsu-Shinohara, M.
Right arrow Articles by Shinohara, T.
Agricola
Right arrow Articles by Kanatsu-Shinohara, M.
Right arrow Articles by Shinohara, T.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS