Foreign Gene Expression in the Mouse Testis by Localizedin VivoGene Transfer

Muramatsu, Tatsuo; Shibata, Osamu; Ryoki, Satoru; Ohmori, Yasushige; Okumura, J.

doi:10.1006/bbrc.1997.6361

Cited by 115 publications

(73 citation statements)

References 24 publications

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“…This increase in transfection efficiency 12,13,15 by elevated voltage is accompanied by the adverse effect of testicular shrinking [12][13][14] . The most favorable voltage range seems to be between 30-50 V, guaranteeing small effects upon testicular integrity, a normal sperm quality 16 , a normal mating behavior 17 , the maintenance of offspring production ability [16][17][18][19][20] as well as a sufficient transfection efficacy.…”

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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“…The method has been shown to be effective in electrotransferring plasmid DNA to various tissues: muscles (Aihara and Miyazaki, 1998;Mir et al, 1998a, liver (Heller et al, 1996;Suzuki et al, 1998), skin (Titomirov et al, 1991;Zhang et al, 1996), tumors (Heller et al, 2000;Wells et al, 2000;Heller and Coppola, 2002), mouse testis (Muramatsu et al, 1997(Muramatsu et al, , 1998, and so on (André and Mir, 2004).…”

Section: Introduction Ementioning

confidence: 99%

Electrophoretic Component of Electric Pulses Determines the Efficacy of In Vivo DNA Electrotransfer

et al. 2005

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Efficient DNA electrotransfer can be achieved with combinations of short high-voltage (HV) and long lowvoltage (LV) pulses that cover two effects of the pulses, namely, target cell electropermeabilization and DNA electrophoresis within the tissue. Because HV and LV can be delivered with a lag up to 3000 sec between them, we considered that it was possible to analyze separately the respective importance of the two types of effects of the electric fields on DNA electrotransfer efficiency. The tibialis cranialis muscles of C57BL/6 mice were injected with plasmid DNA encoding luciferase or green fluorescent protein and then exposed to various combinations of HV and LV pulses. DNA electrotransfer efficacy was determined by measuring luciferase activity in the treated muscles. We found that for effective DNA electrotransfer into skeletal muscles the HV pulse is prerequisite; however, its number and duration do not significantly affect electrotransfer efficacy. DNA electrotransfer efficacy is dependent mainly on the parameters of the LV pulse(s). We report that different LV number, LV individual duration, and LV strength can be used, provided the total duration and field strength result in convenient electrophoretic transport of DNA toward and/or across a permeabilized membrane.

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“…Its advantages, compared with other nonviral gene delivery systems, are that gene expression is drastically increased (2-to 4-log fold), longlasting (months), and very specific and localized . This technique has also been successful in various tissues such as skeletal muscle (Muramatsu et al, 1998;Aihara and Miyazaki, 1999;Gehl and Mir, 1999;Imai and Isaka, 1999;Mathiesen, 1999;Rizzuto et al, 1999;Bettan et al, 2000;Lemieux et al, 2000;Maruyama et al, 2000;Vicat et al, 2000;Lucas and Heller, 2001;Muramatsu et al, 2001), liver (Suzuki et al, 1998;Heller et al, 2000), testis (Muramatsu et al, 1997;Yamazaki et al, 1998;Yamazaki et al, 2000), skin (Johnson et al, 1998;Vanbever and Preat, 1999;Glasspool-Malone et al, 2000), cornea (Oshima et al, 1998;Sakamoto et al, 1999), cardiaovascular (Harrison et al, 1998;Martin et al, 2000) and mammary tumor tissue Wells et al, 2000;Lohr et al, 2001). In particular, gene delivery to skeletal muscle is a promising strategy for the systemic secretion of therapeutic proteins.…”

Section: Introductionmentioning

confidence: 99%

Optimal salt concentration of vehicle for plasmid DNA enhances gene transfer mediated by electroporation

Lee

Cho²,

Jang³

et al. 2002

Exp Mol Med

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In vivo electroporation has emerged as a leading technology for developing nonviral gene therapies, and the various technical parameters governing electroporation efficiency have been optimized by both theoretical and experimental analysis. However, most electroporation parameters focused on the electric conditions and the preferred vehicle for plasmid DNA injections has been normal saline. We hypothesized that salts in vehicle for plasmid DNA must affect the efficiency of DNA transfer because cations would alter ionic atmosphere, ionic strength, and conductivity of their medium. Here, we show that half saline (71 mM) is an optimal vehicle for in vivo electroporation of naked DNA in skeletal muscle. With various salt concentrations, two reporter genes, luciferase and β β β β-galactosidase were injected intramuscularly under our optimal electric condition (125 V/cm, 4 pulses x 2 times, 50 ms, 1 Hz). Exact salt concentrations of DNA vehicle were measured by the inductively coupled plasma-atomic emission spectrometer (ICP-AES) and the conductivity change in the tissue induced by the salt in the medium was measured by Low-Frequency (LF) Impedance Analyzer. Luciferase expression increased as cation concentration of vehicle decreased and this result can be visualized by X-Gal staining. However, at lower salt concentration, transfection efficiency was diminished because the hypoosmotic stress and electrical injury by low conductivity induced myofiber damage. At optimal salt concentration (71 mM), we observed a 3-fold average increase in luciferase expression in comparison with the normal saline condition (p < 0.01). These results provide a valuable experimental parameter for in vivo gene therapy mediated by electroporation.

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Foreign Gene Expression in the Mouse Testis by Localizedin VivoGene Transfer

Cited by 115 publications

References 24 publications

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

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

Electrophoretic Component of Electric Pulses Determines the Efficacy of In Vivo DNA Electrotransfer

Optimal salt concentration of vehicle for plasmid DNA enhances gene transfer mediated by electroporation

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