Direct injection of foreign DNA into mouse testis as a possible alternative of sperm‐mediated gene transfer

The aim of this experiment is to transfect spermatogonia, a stem cell for matured spermatozoa, as well as matured sperm with foreign DNA by means of direct DNA injection into a testis, in order to obtain transgenic mice. Circular plasmid pSV2‐cat was co‐precipitated with calcium phosphate and directly injected via a needle into the mouse testis. Southern blot analysis indicated that the introduced DNA was present as a circular form at least up to 7 days after injection, but was undetectable upon autopsy at 28 days after injection. This suggests that spermatogonia as well as other testicular cells may not be stably transfected by the DNA. PCR (polymerase chain reaction) analysis revealed that the introduced DNA could be detectable in the freshly isolated spermatozoa from caput and cauda epididymides as early as 6 hours after the injection. Furthermore, the DNA was also detectable even in the ejaculated spermatozoa isolated from the uteri of females, which had been successfully mated to the male mice injected with the DNA. However, 1‐cell stage eggs that were fertilized by these sperm cells showed no evidence for sperm‐mediated gene transfer.