title: Adeno-associated virus-mediated delivery of genes to mouse spermatogonial stem cells.
abstract: Spermatogenesis is a complicated process that originates from spermatogonial stem cells (SSCs), which have self-renewal activity. Because SSCs are the only stem cells in the body that transmit genetic information to the next generation, they are an attractive target for germline modification. Although several virus vectors have been successfully used to transduce SSCs, cell toxicity or insertional mutagenesis of the transgene has limited their usage. Adeno-associated virus (AAV) is unique among virus vectors because of its target specificity and low toxicity in somatic cells, and clinical trials have shown that it has promise for gene therapy. However, there are conflicting reports on the possibility of germline integration of AAV into the genome of male germ cells, including SSCs. Here, we examined the usefulness of AAV vectors for exploring germline gene modification in SSCs. AAV1 infected cultured SSCs without apparent toxicity. Moreover, SSCs that were infected in fresh testis cells generated normal appearing spermatogenic colonies after spermatogonial transplantation. A microinsemination experiment produced offspring that underwent excision of the floxed target gene by AAV1-mediated Cre expression. Analysis of the offspring DNA showed no evidence of AAV integration, suggesting a low risk of germline integration by AAV infection. Although more extensive experiments are required to assess the risk of germline integration, our results show that AAV1 is useful for genetic manipulation of SSCs, and gene transduction by AAV will provide a useful approach to overcome potential problems associated with previous virus vector-mediated gene transduction.
authors.affiliation: Department of Molecular Genetics, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Watanabe
authors.affiliation: Department of Molecular Genetics, Graduate School of Medicine, Kyoto University, Kyoto, Japan.|Japan Science and Technology Agency, PRESTO, Kyoto, Japan.
authors.forename: Mito
authors.initials: M
authors.lastname: Kanatsu-Shinohara
authors.affiliation: RIKEN, Bioresource Center, Tsukuba, Japan.
authors.forename: Narumi
authors.initials: N
authors.lastname: Ogonuki
authors.affiliation: RIKEN, Bioresource Center, Tsukuba, Japan.
authors.forename: Shogo
authors.initials: S
authors.lastname: Matoba
authors.affiliation: RIKEN, Bioresource Center, Tsukuba, Japan.
authors.forename: Atsuo
authors.initials: A
authors.lastname: Ogura
authors.affiliation: Department of Molecular Genetics, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Shinohara
mesh_terms: D000256:Adenoviridae; D000072956:Adult Germline Stem Cells; D000818:Animals; D018014:Gene Transfer Techniques; D008297:Male; D008815:Mice, Inbred Strains; D065288:Serogroup
keywords: Adeno-associated virus; Germline stem cell; Spermatogonial stem cell; gene transduction