Direct injection of foreign DNA into mouse testis as a possible in vivo gene transfer system via epididymal spermatozoa

We have attempted to transfect testicular spermatozoa with plasmid DNA by d irect injection into testes to obtain transgenic animals [this technique wa s thus termed "testis-mediated gene transfer (TMGT)"]. When injected males were mated with superovulated females 2 and 3 days after injection, (i) hig h efficiencies (more than 50%) of gene transmission were achieved in the mi dgestational F0 fetuses, (ii) the copy number of plasmid DNA in the fetuses was estimated to be less than I copy per diploid cell, and (iii) overt gen e expression was not found in these fetuses. These findings suggest the pos sibility that plasmid DNA introduced into a testis is rapidly transported t o the epididymis and then incorporated by epididymal spermatozoa. The purpo se of this study was to elucidate the mechanism of TMGT by introducing tryp an blue (TB) or Hoechst 33342 directly into testis. We found that TB is tra nsported to the ducts of the caput epididymis via rete testis within I min after testis injection, and TB reached the corpus and cauda epididymis with in 2-4 days after injection. Staining of spermatozoa isolated from any port ion of epididymis was observed 4 days after injection of a solution contain ing Hoechst 33342. Injection of enhanced green fluorescent protein (EGFP) e xpression vector/liposome complex into testis resulted in transfection of e pithelial cells of epididymal ducts facing the lumen, although the transfec tion efficiency appeared to be low. In vivo electroporation toward the capu t epididymis immediately after injection of EGFP expression vector into a t estis greatly improved the uptake of foreign DNA by the epididymal epitheli al cells. PCR analysis using spermatozoa isolated from corpus and cauda epi didymis 4 days after injection of a DNA/liposome complex into testis reveal ed exogenous DNA in these spermatozoa even after treatment with DNase I. Th ese findings indicate that exogenous DNA introduced into tesits is rapidly transported to epididymal ducts via the rete testis and efferent ducts, and then incorporated by epithelial cells of epididymis and epididymal spermat ozoa. Mol. Reprod. Dev. 61: 49-56, 2002. (C) 2002 Wiley-Liss, Inc.