小鼠精原干细胞转导与移植的初步研究
|
摘要
目的：通过比较脂质体转染法与慢病毒介导法转导小鼠精原干细胞,确立适宜小鼠精原干细胞的体外转基因操作方法,获得GFP-阳性精原干细胞后通过睾丸输出小管显微注射法进行小鼠精原干细胞移植,以期观察GFP阳性小鼠精原干细胞在受体小鼠睾丸中的存活情况。
方法：利用本所已建立的ICR品系新生小鼠精原干细胞系,比较了脂质体介导法和慢病毒介导法转染精原干细胞的效率。确立了适宜的方法,即通过脂质体介导,载体质粒与慢病毒包装质粒pCMV、pMD. G共转染293FT细胞,制备慢病毒(Lentivirus-GFP),检测病毒滴度。通过慢病毒将带有绿色荧光蛋白(GFP)标记的病毒表达载体转导入小鼠精原干细胞内,使其发出绿色荧光。收集GFP阳性精原干细胞后进行体外培养与扩增,于移植前消化成单细胞悬液,经睾丸输出小管显微注射技术,对白消安腹腔注射处理的不育小鼠行同种异体移植,观察GFP阳性精原干细胞在受体小鼠睾丸内的存活情况。
结果：慢病毒可有效转导小鼠精原干细胞,较普通脂质体转染法获得更多GFP阳性细胞。移植术后30天荧光解剖显微镜下可见睾丸组织有散在光点,但未见曲细精管发出明显绿色荧光,睾丸组织冰冻切片内可见少量绿色荧光蛋白表达阳性的精原干细胞沿着曲细精管基底膜生长增殖。
结论：
1、证实了慢病毒介导法是一种适宜小鼠精原干细胞的转导方法。
2、建立了白消安腹腔注射法不育受体小鼠模型。
3、成功运用输出小管显微注射技术完成精原干细胞移植。
4、初步探讨了ICR品系新生小鼠精原干细胞移植术后细胞存活情况。
Objective:By comparing the lipofection method with lentivirus-mediated transduction of mouse spermatogonial stem cells,to establish appropriate method of mice spermatogonial stem cells genetically modified in vitro,to obtain GFP-positive spermatogonial stem cells. Establish the platform of mice spermatogonial stem cell transplantation technology through the method of testicular efferent duct microscopy injection, in order to observe the survival situation of GFP-positive spermatogonial stem cells in the recipient mice testes.
Method:Through the lentivirus, virus vector marked with the green fluorescent protein (GFP)gene is transducted into the spermatogonial stem cells which established before from newborn male ICR mice, and make it radiate Green fluorescent. Cultured and proliferated in vitro after collecting the GFP-positive cells, and then digested into cell suspension. Take an allotransplantation via efferent ductules of testis microinjection on the sterile mice which has been intraperitoneal injected with busulfan. Finally, observe whether or not the GFP-positive spermatogonial stem cell survival in the mouse testis and the process of spermatogenesis.
Results:lentivirus transduction could be a efficient method to obtain GFP-positive spermatogonial stem cells.30 days after transplantation,the testis with scattered points of light could be seen under fluorescent dissecting microscope, however, no significant green fluorescent issued from seminiferous tubule, and a few of spermatogonial stem cells expressed GFP growing and proliferating along with the basement membrane of contorted seminiferous tubules in the testis frozen sections.
Conclusions:
1. It confirmed that Lentivirus-mediated is a suitable method for mice spermatogonial stem cell transduction.
2. Established the mouse infertility receptor model by intraperitoneal injection of busulfan.
3.Successfully completed the spermatogonial stem cell transplantation by using the efferent ductules of testis microinjection.
4. Preliminarily investigated in the newborn ICR mice spermatogonial stem cell survival situation after cell transplantation.
方法：利用本所已建立的ICR品系新生小鼠精原干细胞系,比较了脂质体介导法和慢病毒介导法转染精原干细胞的效率。确立了适宜的方法,即通过脂质体介导,载体质粒与慢病毒包装质粒pCMV、pMD. G共转染293FT细胞,制备慢病毒(Lentivirus-GFP),检测病毒滴度。通过慢病毒将带有绿色荧光蛋白(GFP)标记的病毒表达载体转导入小鼠精原干细胞内,使其发出绿色荧光。收集GFP阳性精原干细胞后进行体外培养与扩增,于移植前消化成单细胞悬液,经睾丸输出小管显微注射技术,对白消安腹腔注射处理的不育小鼠行同种异体移植,观察GFP阳性精原干细胞在受体小鼠睾丸内的存活情况。
结果：慢病毒可有效转导小鼠精原干细胞,较普通脂质体转染法获得更多GFP阳性细胞。移植术后30天荧光解剖显微镜下可见睾丸组织有散在光点,但未见曲细精管发出明显绿色荧光,睾丸组织冰冻切片内可见少量绿色荧光蛋白表达阳性的精原干细胞沿着曲细精管基底膜生长增殖。
结论：
1、证实了慢病毒介导法是一种适宜小鼠精原干细胞的转导方法。
2、建立了白消安腹腔注射法不育受体小鼠模型。
3、成功运用输出小管显微注射技术完成精原干细胞移植。
4、初步探讨了ICR品系新生小鼠精原干细胞移植术后细胞存活情况。
Objective:By comparing the lipofection method with lentivirus-mediated transduction of mouse spermatogonial stem cells,to establish appropriate method of mice spermatogonial stem cells genetically modified in vitro,to obtain GFP-positive spermatogonial stem cells. Establish the platform of mice spermatogonial stem cell transplantation technology through the method of testicular efferent duct microscopy injection, in order to observe the survival situation of GFP-positive spermatogonial stem cells in the recipient mice testes.
Method:Through the lentivirus, virus vector marked with the green fluorescent protein (GFP)gene is transducted into the spermatogonial stem cells which established before from newborn male ICR mice, and make it radiate Green fluorescent. Cultured and proliferated in vitro after collecting the GFP-positive cells, and then digested into cell suspension. Take an allotransplantation via efferent ductules of testis microinjection on the sterile mice which has been intraperitoneal injected with busulfan. Finally, observe whether or not the GFP-positive spermatogonial stem cell survival in the mouse testis and the process of spermatogenesis.
Results:lentivirus transduction could be a efficient method to obtain GFP-positive spermatogonial stem cells.30 days after transplantation,the testis with scattered points of light could be seen under fluorescent dissecting microscope, however, no significant green fluorescent issued from seminiferous tubule, and a few of spermatogonial stem cells expressed GFP growing and proliferating along with the basement membrane of contorted seminiferous tubules in the testis frozen sections.
Conclusions:
1. It confirmed that Lentivirus-mediated is a suitable method for mice spermatogonial stem cell transduction.
2. Established the mouse infertility receptor model by intraperitoneal injection of busulfan.
3.Successfully completed the spermatogonial stem cell transplantation by using the efferent ductules of testis microinjection.
4. Preliminarily investigated in the newborn ICR mice spermatogonial stem cell survival situation after cell transplantation.
引文
[1]Li-Xin Feng, Yali Chen, Luis Dettin, et al, Generation and in Vitro Differentiation of a Spermatogonial Cell Line SCIENCE[J] 2002 VOL 297,19 JULY:392-395
[2]de Rooij DG. Proliferation and differentiation of spermatogonial stem cells. Reproduction 2001,121:347-354.
[3]ANS M. M. VAN PELT, HERMIEN L.et al, Establishment of Cell Lines with Rat Spermatogonial Stem Cell Characteristics. Endocrinology [J]2004143(5):1845-1850
[4]HERKANWAL KHAIRA, DEREK MCLEAN, DANA A. OHLet al, Spermatogonial Stem Cell Isolation,Storage, and Transplantation Journal of Andrology[j], July/August 2005 Vol.26, No.4,:442-451
[5]Hiroshi Kubota and Ralph L Brinster. Technology Insight:in vitro culture of spermatogonial stem cells and their potential therapeutic uses. NATURE CLINICAL PRACTICE ENDOCRINOLOGY & METABOLISM,FEBRUARY 2006 VOL 2 NO 2:99-107
[6]DOLORES D. MRUK AND C. YAN CHENG Sertoli-Sertoli and Sertoli-Germ Cell Interactions and Their Significance in Germ Cell Movement in the Seminiferous Epithelium during Spermatogenesis Endocrine Reviews 2004.25(5):747-806
[7]Karen A. L. Tan, Karel De Gendt,et al, The Role of Androgens in Sertoli Cell Proliferation and Functional Maturation: Studies in Mice with Total or Sertoli Cell-Selective Ablation of the Androgen Receptor. Endocrinology2005 146(6):2674-2683
[8]Shinohara, T., Avarbock, M. R., and Brinster, R. L. Functional analysis of spermatogonial stem cells in Steel and cryptorchid infertile mouse models. Dev. Biol.2000,220:401-411.
[9]BRINSTERR L, ZINUMERMANN JW.Spermato-genesis following male germ-celltransplantation FJ3.Proc Nat Acad Sci USA,1994,91(24):11298-11302.
[10]Brinster RL, Nagano M. Spermatogonial stem cell transplantation, eryopreservation and culture[J]. Semin Cell Dev Biol,1998,9(4):401-409.
[11]孙源,范立青,陶科等小鼠精原干细胞体外培养体系的建立,中华男科学杂志[J],2008第14卷第8期,695-700.
[12]BUOM-YONG RYU, KYLE E. ORWIG, JON M. OATLEY et al. Efficient Generation of Transgenic Rats Through the Male Germline Using Lentiviral
Transduction and Transplantation of Spermatogonial Stem Cells Journal of Andrology[J] 2007, Vol.28, No.2,
[13]谭碧君,慢病毒载体在制备转基因动物中的最新进展,上海畜牧兽医通讯2010年第1期.18-19
[14]Nagano M,Shinohara T,Mary R. Retrovirus2mediated gene delivery into male germline stemcells. FEBS Letters,2000,475:7-10.
[15]Lois C,Elizabeth J. Hong Shirley P,et al. Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors (J) Science,2002,295:868-872.
[16]Kyle E Orwig,Mary R Avarbock,Ralph L Brinster. Retrovirus mediated modification of male germline stem cells in rats.Biology of Reproduction,2002,67:874-879.
[17]Shinohara MK, Toyokuni S, Shinohara T. Transgenic miceproduced by retroviral transduction of male germ line stem cells in vivo. Biol Reprod,2004,71(4): 1202-7
[18]Hamra F K. Production of transgenic rats by lentiviral transduction of male germ-line stem cells (J). Proc Natl Acad Sci USA,2002,99(23):14931-14936.
[19]Khaira H, McLean D, OhlDA, et al. Spermatogonial stem cell isolation, storage, and transp lantation [J]. J Androl,2005,26(4):442-450.
[20]Hibbitt O, Coward K, Kubota H, et al. In vivo gene transfer by electroporation allows expression of a fluorescent transgene in hamster testis and epididymal sperm and has no adverse effects upon testicular integrity or sperm quality. BiolReprod,2006,74(1):95-101
[21]Honaramooz A, Megee S, Zeng W, et al. Adeno-associated virus (AAV)-mediated transduction of male germ line stem cells results in transgene transmission after germ cell transplantation. FASEB J,2008,22(2):374-82
[22]Yu F, Ding LJ, Sun GB, Sun PX, He XH, Ni LG, Li BC Transgenic sperm produced by electrotransfection and allogeneic transplantation of chicken fetal spermatogonial stem cells. Mol Reprod Dev.2010 Apr;77(4):340-7.
[23]Ogawa T,A rechaga JM, Avarbock MR, et al. Transplantation of testis germinal cells into mouse seminiferous tubules. Int J Dev Biol JT-The International journal of developmental biology,1997,41:111-22.
[24]Shinohara T, Orwig K E, Avarbock M R et al. Germ Line Stem Cell Competition in Postnatal Mouse Testis. Biol Re2 pro,2002,66(6):1491
[25]Hamra FK, Chapman KM, Nguyen DM,et al, Self renewal, expansion, and transfection of rat spermatogonial stem cells in culture.Proc Natl Acad Sci U S A. 2005 Nov 29;102(48):17430-5. Epub 2005 Nov 17.
[26]张茨王玲珑金化民等白消安诱导小鼠无精子症模型,医学新知杂志[J]2003年第13卷第4期.
[27]Orwig KE,Avarbock MR,Brinster RL.Retrovirus mediated modification of male germline stem cells in rats.Biol Report,2002,67:874-879.
[28]Clouthier DE,Avarbock MR,Maika SD,et al.Rat spermatogenesis in mouse testes following spermatogonial stem cell transplantation.Nature,1996,381(6581):418-421.
[29]De Miguel MP,Donovan PJ.Determinants of retroviral mediated gene delivery to mouse spermatogonia.Biol Reprod,2003,68(3):860-866.
[30]Kanatsu-Shinohara, M. Miki, H.Inoue, K., Ogonuki, N. Toyokuni, S. Ogura, A., and Shinohara, T. Long-term culture of mouse male germline stem cells under serum or feeder-free conditions. Biol. Reprod 2005,72:985-991.
[31]Guan K, Nayernia K, Maier LS, Wagner S, Dressel R, Lee JH, Nolte J, Wolf F, Li M, Engel W, Hasenfuss G.Pluripotency of spermatogonial stem cells from adult mouse testis. Nature 2006,440:1199-1203.
[1]M.C. Nagano, Homing efficiency and proliferation kinetics of male germ line stem cells following transplantation in mice,Biol Reprod [J]69(2)2003,701-707.
[2]M.Kanatsu-Shinohara et al., Clonal origin of germ cell colonies after spermatogonial transplantation in mice, Biol Reprod [J]75(1) 2006,68-74.
[3]Matsui Y, et al.Primordial germ cells contain subpopulations that have greater ability to develop into pluripotential stem cells. Dev Growth Differ. [J] 2009 Sep;51(7):657-67
[4]Matzuk MM.Germ-line immortality.Proc Natl Acad Sci USA 2004,101:16395-16396
[5]BRINSTER R L, ZINUMERMANN JW.Spermato-genesis following male germ-celltransplantation FJ3.Proc Nat Acad Sci USA,1994,91(24):11298-11 302.
[6]NAGANO M,SHINOHARA T,AVARBOCK MR,etal.Retrovirus-mediated gene delivery into male germ line stem cells[J].FEBS Letters,2000,475:7-10.
[7]HAMRA FK,GATLINJ,CHAPMANKM,et al.Production of trans-genie rats by lentiviral transduction of male germ-line stem cells[J]Proc Natl Acad Sei USA,2002,99:14931-14936.
[8]杨兴坤、黄天华、谢庆东等。脂质体介导的精原细胞基因转移[J].汕头大学学报,2006,18(4)：277-280.
[9]Chang LJ, Avarbock MR, Brinster RL, et al. Efficient generation of transgenic rats through the male germline usinglentiviral transduction and transplantation of spermatogonial stem cells. J Androl,2007,28(2):353-60
[10]ClouthierDE, AvarbockMR, Maika SD, et al. Rat spermato genesis in mouse testis[J]. Nature,1996,381 (6581):
[11]scIlla S,Rosiepen G.Weinbauer GF.et al.Germ cel transfer into rat,bovine,monkey and humantestes. Hum Reprod.1999.14:144.150.
[12]Nagano M,McCarrey JR,Brinster RL.Primate spemmtogonial stem cells colonize mouse testes.Biol Reprod,2001.64:1409.1416
[13][13]Kaul G, Kaur J, Rafeeqi TA. Ultrasound Guided Transplantation of Enriched and Cryopreserved Spermatogonial Cell Suspension in Goats. Reprod Domest Anim.2009 Dec 4.
[14]Kubota H, Avarbock MR, Schmidt JA, Brinster RL Spermatogonial stem cells derived from infertile Wv/Wv mice self-renew in vitro and generate progeny following transplantation. Biol Reprod.2009 Aug;81(2):293-301.Epub 2009 Apr 15.
[15]Shen F, Zhang C, Zheng H, et al, Wang L.Long-term culture and transplantation of spermatogonial stem cells from BALB/c mice.Cells Tissues Organs[J].2010;191(5):372-81. Epub 2010 Jan 14
[16]Kanatsu-Shinohara M, Takashima S, Shinohara T.Transmission distortion by loss of p21 or p27 cyclin-dependent kinase inhibitors following competitive spermatogonial transplantation.Proc Natl Acad Sci U S A.2010 Apr 6;107(14):6210-5. Epub 2010 Mar 22.
[17]Yu F, Ding LJ, Sun GB, Sun PX, He XH, Ni LG, Li BC Transgenic sperm produced by electrotransfection and allogeneic transplantation of chicken fetal spermatogonial stem cells. Mol Reprod Dev.2010 Apr;77(4):340-7.
[18]Ogawa T,A rechaga JM, Avarbock MR, et al. Transplantation of testis germinal cells into mouse seminiferous tubules. Int J Dev Biol JT-The International journal of developmental biology,1997,41:111-22.
[2]de Rooij DG. Proliferation and differentiation of spermatogonial stem cells. Reproduction 2001,121:347-354.
[3]ANS M. M. VAN PELT, HERMIEN L.et al, Establishment of Cell Lines with Rat Spermatogonial Stem Cell Characteristics. Endocrinology [J]2004143(5):1845-1850
[4]HERKANWAL KHAIRA, DEREK MCLEAN, DANA A. OHLet al, Spermatogonial Stem Cell Isolation,Storage, and Transplantation Journal of Andrology[j], July/August 2005 Vol.26, No.4,:442-451
[5]Hiroshi Kubota and Ralph L Brinster. Technology Insight:in vitro culture of spermatogonial stem cells and their potential therapeutic uses. NATURE CLINICAL PRACTICE ENDOCRINOLOGY & METABOLISM,FEBRUARY 2006 VOL 2 NO 2:99-107
[6]DOLORES D. MRUK AND C. YAN CHENG Sertoli-Sertoli and Sertoli-Germ Cell Interactions and Their Significance in Germ Cell Movement in the Seminiferous Epithelium during Spermatogenesis Endocrine Reviews 2004.25(5):747-806
[7]Karen A. L. Tan, Karel De Gendt,et al, The Role of Androgens in Sertoli Cell Proliferation and Functional Maturation: Studies in Mice with Total or Sertoli Cell-Selective Ablation of the Androgen Receptor. Endocrinology2005 146(6):2674-2683
[8]Shinohara, T., Avarbock, M. R., and Brinster, R. L. Functional analysis of spermatogonial stem cells in Steel and cryptorchid infertile mouse models. Dev. Biol.2000,220:401-411.
[9]BRINSTERR L, ZINUMERMANN JW.Spermato-genesis following male germ-celltransplantation FJ3.Proc Nat Acad Sci USA,1994,91(24):11298-11302.
[10]Brinster RL, Nagano M. Spermatogonial stem cell transplantation, eryopreservation and culture[J]. Semin Cell Dev Biol,1998,9(4):401-409.
[11]孙源,范立青,陶科等小鼠精原干细胞体外培养体系的建立,中华男科学杂志[J],2008第14卷第8期,695-700.
[12]BUOM-YONG RYU, KYLE E. ORWIG, JON M. OATLEY et al. Efficient Generation of Transgenic Rats Through the Male Germline Using Lentiviral
Transduction and Transplantation of Spermatogonial Stem Cells Journal of Andrology[J] 2007, Vol.28, No.2,
[13]谭碧君,慢病毒载体在制备转基因动物中的最新进展,上海畜牧兽医通讯2010年第1期.18-19
[14]Nagano M,Shinohara T,Mary R. Retrovirus2mediated gene delivery into male germline stemcells. FEBS Letters,2000,475:7-10.
[15]Lois C,Elizabeth J. Hong Shirley P,et al. Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors (J) Science,2002,295:868-872.
[16]Kyle E Orwig,Mary R Avarbock,Ralph L Brinster. Retrovirus mediated modification of male germline stem cells in rats.Biology of Reproduction,2002,67:874-879.
[17]Shinohara MK, Toyokuni S, Shinohara T. Transgenic miceproduced by retroviral transduction of male germ line stem cells in vivo. Biol Reprod,2004,71(4): 1202-7
[18]Hamra F K. Production of transgenic rats by lentiviral transduction of male germ-line stem cells (J). Proc Natl Acad Sci USA,2002,99(23):14931-14936.
[19]Khaira H, McLean D, OhlDA, et al. Spermatogonial stem cell isolation, storage, and transp lantation [J]. J Androl,2005,26(4):442-450.
[20]Hibbitt O, Coward K, Kubota H, et al. In vivo gene transfer by electroporation allows expression of a fluorescent transgene in hamster testis and epididymal sperm and has no adverse effects upon testicular integrity or sperm quality. BiolReprod,2006,74(1):95-101
[21]Honaramooz A, Megee S, Zeng W, et al. Adeno-associated virus (AAV)-mediated transduction of male germ line stem cells results in transgene transmission after germ cell transplantation. FASEB J,2008,22(2):374-82
[22]Yu F, Ding LJ, Sun GB, Sun PX, He XH, Ni LG, Li BC Transgenic sperm produced by electrotransfection and allogeneic transplantation of chicken fetal spermatogonial stem cells. Mol Reprod Dev.2010 Apr;77(4):340-7.
[23]Ogawa T,A rechaga JM, Avarbock MR, et al. Transplantation of testis germinal cells into mouse seminiferous tubules. Int J Dev Biol JT-The International journal of developmental biology,1997,41:111-22.
[24]Shinohara T, Orwig K E, Avarbock M R et al. Germ Line Stem Cell Competition in Postnatal Mouse Testis. Biol Re2 pro,2002,66(6):1491
[25]Hamra FK, Chapman KM, Nguyen DM,et al, Self renewal, expansion, and transfection of rat spermatogonial stem cells in culture.Proc Natl Acad Sci U S A. 2005 Nov 29;102(48):17430-5. Epub 2005 Nov 17.
[26]张茨王玲珑金化民等白消安诱导小鼠无精子症模型,医学新知杂志[J]2003年第13卷第4期.
[27]Orwig KE,Avarbock MR,Brinster RL.Retrovirus mediated modification of male germline stem cells in rats.Biol Report,2002,67:874-879.
[28]Clouthier DE,Avarbock MR,Maika SD,et al.Rat spermatogenesis in mouse testes following spermatogonial stem cell transplantation.Nature,1996,381(6581):418-421.
[29]De Miguel MP,Donovan PJ.Determinants of retroviral mediated gene delivery to mouse spermatogonia.Biol Reprod,2003,68(3):860-866.
[30]Kanatsu-Shinohara, M. Miki, H.Inoue, K., Ogonuki, N. Toyokuni, S. Ogura, A., and Shinohara, T. Long-term culture of mouse male germline stem cells under serum or feeder-free conditions. Biol. Reprod 2005,72:985-991.
[31]Guan K, Nayernia K, Maier LS, Wagner S, Dressel R, Lee JH, Nolte J, Wolf F, Li M, Engel W, Hasenfuss G.Pluripotency of spermatogonial stem cells from adult mouse testis. Nature 2006,440:1199-1203.
[1]M.C. Nagano, Homing efficiency and proliferation kinetics of male germ line stem cells following transplantation in mice,Biol Reprod [J]69(2)2003,701-707.
[2]M.Kanatsu-Shinohara et al., Clonal origin of germ cell colonies after spermatogonial transplantation in mice, Biol Reprod [J]75(1) 2006,68-74.
[3]Matsui Y, et al.Primordial germ cells contain subpopulations that have greater ability to develop into pluripotential stem cells. Dev Growth Differ. [J] 2009 Sep;51(7):657-67
[4]Matzuk MM.Germ-line immortality.Proc Natl Acad Sci USA 2004,101:16395-16396
[5]BRINSTER R L, ZINUMERMANN JW.Spermato-genesis following male germ-celltransplantation FJ3.Proc Nat Acad Sci USA,1994,91(24):11298-11 302.
[6]NAGANO M,SHINOHARA T,AVARBOCK MR,etal.Retrovirus-mediated gene delivery into male germ line stem cells[J].FEBS Letters,2000,475:7-10.
[7]HAMRA FK,GATLINJ,CHAPMANKM,et al.Production of trans-genie rats by lentiviral transduction of male germ-line stem cells[J]Proc Natl Acad Sei USA,2002,99:14931-14936.
[8]杨兴坤、黄天华、谢庆东等。脂质体介导的精原细胞基因转移[J].汕头大学学报,2006,18(4)：277-280.
[9]Chang LJ, Avarbock MR, Brinster RL, et al. Efficient generation of transgenic rats through the male germline usinglentiviral transduction and transplantation of spermatogonial stem cells. J Androl,2007,28(2):353-60
[10]ClouthierDE, AvarbockMR, Maika SD, et al. Rat spermato genesis in mouse testis[J]. Nature,1996,381 (6581):
[11]scIlla S,Rosiepen G.Weinbauer GF.et al.Germ cel transfer into rat,bovine,monkey and humantestes. Hum Reprod.1999.14:144.150.
[12]Nagano M,McCarrey JR,Brinster RL.Primate spemmtogonial stem cells colonize mouse testes.Biol Reprod,2001.64:1409.1416
[13][13]Kaul G, Kaur J, Rafeeqi TA. Ultrasound Guided Transplantation of Enriched and Cryopreserved Spermatogonial Cell Suspension in Goats. Reprod Domest Anim.2009 Dec 4.
[14]Kubota H, Avarbock MR, Schmidt JA, Brinster RL Spermatogonial stem cells derived from infertile Wv/Wv mice self-renew in vitro and generate progeny following transplantation. Biol Reprod.2009 Aug;81(2):293-301.Epub 2009 Apr 15.
[15]Shen F, Zhang C, Zheng H, et al, Wang L.Long-term culture and transplantation of spermatogonial stem cells from BALB/c mice.Cells Tissues Organs[J].2010;191(5):372-81. Epub 2010 Jan 14
[16]Kanatsu-Shinohara M, Takashima S, Shinohara T.Transmission distortion by loss of p21 or p27 cyclin-dependent kinase inhibitors following competitive spermatogonial transplantation.Proc Natl Acad Sci U S A.2010 Apr 6;107(14):6210-5. Epub 2010 Mar 22.
[17]Yu F, Ding LJ, Sun GB, Sun PX, He XH, Ni LG, Li BC Transgenic sperm produced by electrotransfection and allogeneic transplantation of chicken fetal spermatogonial stem cells. Mol Reprod Dev.2010 Apr;77(4):340-7.
[18]Ogawa T,A rechaga JM, Avarbock MR, et al. Transplantation of testis germinal cells into mouse seminiferous tubules. Int J Dev Biol JT-The International journal of developmental biology,1997,41:111-22.