Detection of transgene in progeny at different developmental stages following testis-mediated gene transfer

We recently reported that exogenous DNA injected into testis as a liposome complex can be transferred into the egg via sperm by natural mating and int egrated in the genome (testis-mediated gene transfer: TMGT). Here, we studi ed the efficiency of each of the several liposomes in associating foreign D NA with sperm, the expression of an introduced gene in early embryos, and t he presence of the DNA in fetuses and pups at different ages. The CMV/beta -actin/ EGFP fusion gene, encapsulated with different liposomes, was inject ed into rat testis, and spermatozoa in the cauda epididymis were obtained 1 , 4, and 14 days after injection. We tested each of the 8 liposomes, and fo und that only 2, DMRIE-C and SuperFect, led to the detection of foreign DNA on all of the days examined, with relatively higher ratios of rats having positive sperm. By means of TMGT using either of those two liposomes, more than 80% of morula-stage embryos expressed EGFP, as observed by fluorescenc e microscopy. Then we detected introduced DNA in the progeny by PCR and Sou thern dot blot, and found that the ratio of animals carrying the foreign DN A decreased as they developed, and that only a part of postpartum progeny w ere foreign-DNA-positive with high incidence of mosaicism. These results su ggest that, although, the success rate is still limited, foreign DNA could be integrated into the genome of the progeny by TMGT at least under specifi c experimental conditions, the efficiency of which depends largely on the c haracteristics of the liposome. The results also suggest that TMGT could be applicable to fetal gene therapy as well as to the generation of transgeni c animals. Mol. Reprod. Dev. 60: 196-201, 2001. (C) 2001 Wiley-Liss, Inc.