Male germ cell transplantation: an experimental approach with a clinical perspective

Schlatt, Stefan; Schönfeldt, Viktoria von; Schepers, Andreas G.

doi:10.1258/0007142001903409

Cited by 30 publications

(8 citation statements)

References 32 publications

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“…The first one is the microinjection into seminiferous tubules with the preparation of the injection capillary. This technique has been commonly implicated in germ/stem cell transplantation either from transgenic animals to the recipient 7 or in the context of xenografting 8 . The second method is the electroporation transfection capable of inducing transient pores in the plasma membrane as well as a directed bulk flow of charged molecules like plasmids, ensuring entrance into certain cells or rather tissues.…”

Section: Discussionmentioning

confidence: 99%

<em>In Vivo</em> Microinjection and Electroporation of Mouse Testis

Michaelis

Sobczak

Weitzel

2014

JoVE

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This video and article contribution gives a comprehensive description of microinjection and electroporation of mouse testis in vivo. This particular transfection technique for testicular mouse cells allows the study of unique processes in spermatogenesis.The following protocol focuses on transfection of testicular mouse cells with plasmid constructs. Specifically, we used the reporter vector pEGFP-C1, which expresses enhanced green fluorescent protein (eGFP) and also the pDsRed2-N1 vector expressing red fluorescent protein (DsRed2). Both encoded reporter genes were under the control of the human cytomegalovirus immediate-early promoter (CMV).For performing gene transfer into mouse testes, the reporter plasmid constructs are injected into testes of living mice. To that end, the testis of an anaesthetized animal is exposed and the site of microinjection is prepared. Our preferred place of injection is the efferent duct, with the ultimately connected rete testis as the anatomical transport route of the spermatozoa between the testis and the epididymis. In this way, the filling of the seminiferous tubules after microinjection is excellently managed and controlled due to the use of stained DNA solutions. After observing a sufficient filling of the testis by its colored tubule structure, the organ is electroporated. This enables the transfer of the DNA solution into the testicular cells. Following 3 days of incubation, the testis is removed and investigated under the microscope for green or red fluorescence, illustrating transfection success.Generally, this protocol can be employed for delivering DNA-or RNA-constructs into living mouse testis in order to (over)express or knock down genes, facilitating in vivo gene function analysis. Furthermore, it is suitable for studying reporter constructs or putative gene regulatory elements. Thus, the main advantages of the electroporation technique are fast performance in combination with low effort as well as the moderate technical equipment and skills required compared to alternative techniques. Video LinkThe video component of this article can be found at

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Section: Discussionmentioning

confidence: 99%

<em>In Vivo</em> Microinjection and Electroporation of Mouse Testis

Michaelis

Sobczak

Weitzel

2014

JoVE

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“…Briefly, 40 mg/kg of 20 mg/mL DMSO-Busulfan was injected intraperitoneally into each of 20 cocks to eliminate endogenous SSCs as described [10][11][12][13] . Four weeks later, 2 mL (1×10 6 /mL) of transfected SSCs suspension was injected into each testis of cocks with the same method as TMGT.…”

Section: Ssc Implantationmentioning

confidence: 99%

Efficient generation of transgenic chickens using the spermatogonial stem cells in vivo and ex vivo transfection

Sun

et al. 2008

SCI CHINA SER C

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The highly efficient novel methods to produce transgenic chickens were established by directly injecting the recombinant plasmid containing green fluorescent protein (GFP) gene into the cock's testis termed as testis-medianted gene transfer (TMGT), and transplanting transfected spermatogonial stem cells (TTSSCs). For the TMGT approach, four dosages of pEGFP-N1 DNA/cationic polymer complex were injected intratesticularly. The results showed: (1) 48 h after the injection, the percentages of testis cells expressing GFP were 4.0%, 8.7%, 10.2% and 13.6% in the 50, 100, 150 and 200 microg/mL group, respectively. The difference from the four dosage groups was significant (P<0.05). On day 25 after the injection, a dosage-dependent and time-dependent increase in the number of transgenic sperm was observed. The percentages of gene expression reached the summit and became stable from day 70 to 160, being 12.7%, 12.8%, 15.9% and 19.1%, respectively. The difference from the four dosage groups was also significant (P<0.05). (2) 70 d after the injection, strong green fluorescent could be observed in the seminiferous tubules by whole-mount in-situ hybridization. (3) 70 d after the injection, the semen was collected and used to artificially inseminate wild-type females. The blastoderms of F(1) and F(2) transgenic chicken expressed GFP were 56.2% (254/452) and 53.2% (275/517), respectively. The detection of polymerase chain reaction (PCR) of F(1) and F(2) transgenic chicken blood genomic DNA showed that 56.5% (3/23) of F(1) and 52.9% (9/17) of F(2) were positive. Southern blot showed GFP DNA was inserted in their genomic DNAs. (4) Frozen whole mount tissue sections of F(1) and F(2) transgenic chicken liver, heart, kidney and muscle showed that the rates of green fluorescent positive were between 50.0% and 66.7%. (5) With the TTSSCs method, SSCs ex vivo transfected with GFP were transplanted into recipient roosters whose endogenic SSCs had been resoluted. The donor SSCs settled and GFP expression became readily detectable in the frozen whole mount tissue sections of recepient testes. Moreover, sperms carrying GFP could be produced normally. The results of artificially inseminating wild-type females with these sperms showed 12.5% (8/64) of offspring embryo expressed GFP and 11.1% (2/18) hatched chicks were tested transgenic. Our data therefore suggest TMGT and TTSSCs are the feasible methods for the generation of transgenic chickens.

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“…Germ cell transplantation has become an established experimental tool over the last few years [11][12][13]. Germ cell suspensions obtained after digestion of the testis of donor animals are transferred into the recipient's testicular tubules by microinjection via the rete testis [14].…”

Section: Introductionmentioning

confidence: 99%

Transplantation of Wild-Type Spermatogonia Leads to Complete Spermatogenesis in Testes of Cyclic 3′,5′-Adenosine Monophosphate Response Element Modulator-Deficient Mice1

Wistuba¹,

Schlatt²,

Cantauw³

et al. 2002

Biology of Reproduction

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The cAMP response element modulator (CREM) gene encodes transcription factors that are highly expressed in spermatids. A deficiency of the CREM gene leads to male infertility in mice due to round spermatid maturation arrest. However, CREM is also expressed in testicular Sertoli cells. We investigated whether CREM deficiency affects the germ line alone or whether the testicular environment is also dependent on CREM function. We examined the restoration of donor-derived spermatogenesis in CREM-deficient testes after transfer of wild-type spermatogonia (16 animals) and after transplantation of germ cells from CREM-deficient or heterozygous donors into spermatogenic tubules of wild-type hosts (16 and 12 animals). Six wk after endogenous spermatogenesis had been depleted by busulphan treatment, spermatogonia were transferred via the rete testis. Production of donor-derived germ cells in the deficient recipients was confirmed by testicular histology and polymerase chain reaction (PCR) analysis of testis fragments 7 and 13 wk after germ cell transfer. Sperm with donor genotype, as detected by PCR, also were flushed from the epididymis. Germ cell transfer using heterozygous donors was also successful. CREM-deficient germ cells largely failed to colonize wild-type recipient testes. According to these findings, germ cell differentiation is dependent on CREM function. The testicular environment of CREM-deficient mice is not essentially affected and is able to support complete spermatogenesis in the presence of wild-type germ cells.

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Male germ cell transplantation: an experimental approach with a clinical perspective

Cited by 30 publications

References 32 publications

<em>In Vivo</em> Microinjection and Electroporation of Mouse Testis

<em>In Vivo</em> Microinjection and Electroporation of Mouse Testis

Efficient generation of transgenic chickens using the spermatogonial stem cells in vivo and ex vivo transfection

Transplantation of Wild-Type Spermatogonia Leads to Complete Spermatogenesis in Testes of Cyclic 3′,5′-Adenosine Monophosphate Response Element Modulator-Deficient Mice1

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