Testicular injections

Male mice, F1 hybrids of C57BL/6 J and DBA2J were used for experiments and injections performed as previously reported [25]. The animals were anesthetized by Isoflurane (2-chloro-2-(difluoromethoxy)-1,1,1-trifluoro-ethane), surgical site was sterilized with ethanol and topical application of Betadine. A single incision was made on the ventral skin and body wall about 1.5 cm anterior to the genitals, using sterile surgical scissors under aseptic conditions. The testes were pulled from the scrotal sac holding the fat pad. The volumes of the viral stocks were adjusted with PBS to achieve either 1 X 10⁹ vgs or 1 X 10¹⁰ in 15 μl volume. Each testis was injected 15 μl of the viral suspension using 30G needle syringe. The typical titre we obtain in AAV preparations in laboratory scale is 10¹¹–10¹² viral genomes / ml and the upper limit of the injection volume in the mouse testis capsule is 15 μl. Injection was into intertubular spaces, also known as testis capsule. In a set of animals, left testis served as an uninjected control. The animals were sacrificed after desired period of incubation (3 or 8 days or 4 weeks) post injection and testes were dissected for analysis.