In Vivo Gene Transfer to Mouse Spermatogenic Cells by Deoxyribonucleic Acid Injection into Seminiferous Tubules and Subsequent Electroporation1

Abstract

An in vivo gene transfer technique for living mouse testes was used to develop a novel transient expression assay system for transcriptional regulatory elements of spermatogenic specific genes. The combination of DNA injection into seminiferous tubules and subsequent in vivo electroporation resulted in an efficient and convenient assay system for gene expression during spermatogenesis. The transfer of the firefly luciferase reporting gene driven by the Protamine-1 (Prm-1) enhancer region revealed a significant increase in the activity of the reporter enzyme. Histochemical studies of the transfer of the lacZ gene driven by the Prm-1 enhancer showed specific lacZ expression only in haploid spermatid cells in adult testes, corresponding with the expression pattern of endogenous Prm-1. We were able to detect long-lasting transgene expression in the transfected spermatogenic cells. A group of spermatogenic differentiating cells maintained the transfected lacZ expression after more than 2 mo of transfection, suggesting that spermatogenic stem cells and/or spermatogonia could also incorporate foreign DNA and that the transgene could be transmitted to the progenitor cells derived from a transfected proliferating germ cell.

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

“…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.…”

Adenovirus-mediated gene delivery and in vitro microinsemination produce offspring from infertile male mice

“…Furthermore, genetic modification of Sertoli cells could be used to correct defective Sertoli cells, thus providing a method to treat those cases of male infertility that might be the results of defects in Sertoli cell function. However, although germ cells can be transfected by using several methods (15)(16)(17), there is currently no technique that allows long-term, widespread transgene expression in Sertoli cells (16,18). Adenoviruses have potential as gene therapy vectors in human patients because of their relatively high cloning capacity and amenability to production in high titers (19).…”

Therapeutic Potential of Gene Transfer to Testis; Myth or Reality?

“…As a possible alternative and more effective method for generating transgenic mice, several researchers have attempted testis-mediated gene transfer using an in vivo electroporation method. After injection of exogenous DNA into the testis, the testes are held between a tweezer-type electrode, and square electric pulses are applied several times at 20-50 volts (Muramatsu, 1996(Muramatsu, , 1997Yamazaki, 1998Yamazaki, , 2000Umemoto, 2002. Expression of the exogenous gene was clearly observed in germ cells of seminiferous tubules 48 hours after transfecting the mouse testis by in vivo electroporation (Muramatsu, 1997).…”