In vivo transfection of the mouse vas deferens

Abstract

To explore the possibility that specific characteristics of the epithelium of the male tract can be modified, transfections of the mouse vas deferens have been performed using in vivo injections of cationic DNA/liposome complexes. Gene transfer was done employing the reporter genes pEGFP-C1 encoding Green Fluorescent Protein (GFP) and pCMV-nls-beta encoding the nuclear beta-Galactosidase (beta-Gal). Foreign gene expression reached a maximum of 6.8% in the epithelial cells of the vas after treatment with the nuclear beta-Gal gene construction and of 13.3% after employing the GFP gene construction. Expression of the GFP gene appeared from one week up to three months following injection, and it appeared as patches of modified cells along the epithelium. Results from immunocytochemistry and Western Blotting support the conclusion that transfection of epithelial cells was achieved. We have also transfected the vas using gene constructions that express secretory proteins--specifically, the reporter system pSEAP-control that expresses a secretory form of human placental alkaline phosphatase, and the pGFP-Ctk-37 that expresses a secretion form of GFP. In both cases, the fluids expressed from the transfected vas showed a significant increase of alkaline phosphatase activity after pSEAP transfection and the presence of GFP protein when pGFP-Ctk-37 gene construction was employed. Our results indicate that the vas can be transfected in vivo using liposomes as vectors of foreign genes and that the vas fluid contents can be modified.

Production of transgenic mice expressing tumor virus A under ovarian-specific promoter 1 control using testis-mediated gene transfer

“…Type A spermatogonia are the only immortalized diploid cells that are reproduced in adult male animals, including mice, and they have a high self-renewal capacity and pluripotent potential (34,35). On that basis, and following consideration of the results of the present study along with those of Shen et al (26) and Valenzuela et al (28), it was suggested that the TMGT mechanism may include additional components. Once a foreign DNA and liposome (DOTAP) complex is injected directly into the testis, all cells that have contact with that complex, not only spermatids at different development stages, but also the two types of spermatogonia are considered to be transfected with foreign DNA.…”

“…Sato et al (27) also obtained F2 transgenic-positive animals. However, Shen et al (26) and Valenzuela et al (28) did not identify transgene-positive individuals in an F2 generation. We hypothesize that the transgenic positive rate in F2 offspring is associated with the age at which the F1 parents are mated; the younger the F1 parent, the higher the F2 transgenic positive rate; when the F1 parent ages, the F2 transgenic positive rate decreases.…”

Immunomodulators in tick saliva and their benefits

“…The effect of inflammatory mediators is blocked, perhaps by the same kininase enzyme or other carboxypeptidase (Valenzuela et al, 2002).Tick salivary kininase, for example dipeptidyl carboxypeptidase from I. scapularis tick saliva (Ribeiro and Mather, 1998), hydrolyzed circulating kinins (e.g. bradykinin).…”

“…Besides inhibition of the production of pro-inflammatory cytokines, IL-6, and TNF-α (Leboulle et al, 2002b) and anti-haemostatic effects mentioned above (Prevot et al, 2006), Iris has been shown to suppress T cell proliferation and induces Th2 type immune response. Due to sialotranscriptome work on I. scapularis saliva (Valenzuela et al, 2002), a large number of protease inhibitors was detected in SG, including secreted 12.5 kDa Sialostatin L containing the conserved cystatin domain. It inhibits papain-like cysteine proteases, targeting mainly cathepsin L, C and S. This protein displays an anti-inflammatory effect and reduces the proliferation of cytotoxic T lymphocytes (CTL) (Kotsyfakis et al, 2006), as well as antigen specific CD4 + T cells (Sa-Nunes et al, 2009).…”

Foreign Gene Expression in the Mouse Cauda Epididymis is Regulated by Androgens

“…In the male genital tract in vitro transfections have been developed using epididymal cell lines (Kirchoff et al, 2004;Dufresne et al, 2005;Yu et al, 2006). In the mouse, the vas deferens was in vivo transfected using the GFP gene construction (Valenzuela et al, 2002). In the human an in vitro transfection of the vas was also undertaken (Esponda et al, 2004).…”