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

Abstract

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

MicroRNA‐130a inhibits spermatogenesis by directly targeting androgen receptor in mouse Sertoli cells

“…It has been confirmed previously that the hemisphere of the testis proximal to the injection site shows a higher level of targeting (Michaelis et al, ). Thus, the mice were killed 72 hr after injections and the hemisphere of testis proximal to efferent ductule was removed and processed for qRT‐PCR and Western blot analysis.…”

“…The in vivo testis microinjection was performed as described previously with minor modification (Michaelis, Sobczak, & Weitzel, ). Briefly, 21‐day‐old male C57BL/6 mice were anesthetized with 5% chloral hydrate, and the testes were exposed by a 5 mm midline abdominal incision, agomiR‐130a, and negative control (RiboBio Co. Ltd., Guangzhou, China) were diluted to a final concentration of 40 μM with sterile phosphate‐buffered saline and stored at −20°C.…”

“…The in vivo testis microinjection was performed as described previously with minor modification (Michaelis, Sobczak, & Weitzel, 2014). Briefly, 21-day-old male C57BL/6 mice were anesthetized with 5% chloral hydrate, and the testes were It has been confirmed previously that the hemisphere of the testis proximal to the injection site shows a higher level of targeting (Michaelis et al, 2014). Thus, the mice were killed 72 hr after injections and the hemisphere of testis proximal to efferent ductule was removed and processed for qRT-PCR and Western blot analysis.…”

Decreased Expression of KIFC1 in Human Testes with Globozoospermic Defects

“…Thus, the protein level of the targeting gene will be reduced for a relatively long period. We injected Kifc1 siRNAs into the testes of 3-week-old mice as previously described [35]. We used siRNA rather than dsRNA, which is easily phagocytosed and degraded by Sertoli cells [36].…”

A eficácia do coaching para o aumento de performance

“…Conforme de nição do dicionário Michaelis (2014), habilidade se refere á qualidade de uma pessoa hábil, a capacidade, inteligência e aptidão. Em outras palavras, a maneira como a pessoa demonstra sua competência.…”