Adeno-associated virus-mediated delivery of genes to mouse spermatogonial stem cells
Adeno-associated virus-mediated delivery of genes to mouse spermatogonial stem cells
复制标题
DOI：
10.1095/biolreprod.116.143495
复制
发表时间：
2017-01-01
影响因子：
3.6
通讯作者：
Shinohara, Takashi
中科院分区：
文献类型：
--
作者：
Watanabe, Satoshi;Kanatsu-Shinohara, Mito;Shinohara, Takashi
精子发生是一个复杂的过程，来源于具有自我更新能力的精原干细胞。由于SSCs是体内唯一将遗传信息传递给下一代的干细胞，它们是生殖系改造的一个有吸引力的目标。虽然一些病毒载体已经成功地用于转导SSCs，但转基因的细胞毒性或插入突变限制了它们的使用。腺相关病毒(AAV)因其靶点特异性和对体细胞的低毒作用而在病毒载体中独树一帜，临床试验表明它在基因治疗方面具有广阔的前景。然而，关于AAV的生殖系整合到包括SSCs在内的男性生殖细胞基因组的可能性，有相互矛盾的报道。在这里，我们考察了AAV载体在探索SSCs生殖系基因修饰方面的有用性。AAV1感染培养的SSCs无明显毒性。此外，在新鲜睾丸细胞中感染的SSCs在精原移植后产生了正常的生精集落。一项微授精实验产生了通过AAV1介导的Cre表达来切除目标基因的后代。对后代DNA的分析没有显示AAV整合的证据，这表明AAV感染导致生殖系整合的风险很低。虽然还需要更广泛的实验来评估生殖系整合的风险，但我们的结果表明，AAV1对SSCs的遗传操作是有用的，AAV的基因转导将提供一种有用的方法来克服以前病毒载体介导的基因转导相关的潜在问题。
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.Summary SentenceA new system for spermatogonial stem cell gene transfection is demonstrated.