CRISPR/Cas9 Promotes Functional Study of Testis Specific X-Linked Gene In Vivo.
CRISPR/Cas9 Promotes Functional Study of Testis Specific X-Linked Gene In Vivo.
复制标题
CRISPR/Cas9 促进睾丸特异性 X 连锁基因的体内功能研究
DOI：
10.1371/journal.pone.0143148
复制
发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Huang J
中科院分区：
文献类型：
--
作者：
Li M;Huang R;Jiang X;Chen Y;Zhang Z;Zhang X;Liang P;Zhan S;Cao S;Songyang Z;Huang J
哺乳动物精子发生是一个高度调控的多阶段精子生成过程。由于睾丸特异性基因的体外模型尚不成熟，很难在体外揭示其真实的功能。随着CRISPR/Cas9系统的发展，我们可以快速构建睾丸特异性基因敲除小鼠模型，用于研究精子发生过程。SYCP 3样X连锁2（SLX 2）是一种生殖细胞特异性组分，含有Cor 1结构域，属于XLR（X-linked，lymphocyte regulated）家族。以往的研究表明，SLX 2可能在小鼠精子发生中发挥重要作用，其亚细胞定位和相互作用蛋白。然而，SLX 2在体内的功能仍然是难以捉摸的。在这里，为了研究SLX 2在精子发生中的功能，我们通过使用CRISPR/Cas9系统破坏了Slx 2基因。由于Slx 2是睾丸特异性X连锁基因，我们在第一代获得了基因敲除的雄性小鼠，加快了研究进程。与野生型小鼠相比，Slx 2基因敲除小鼠具有正常的睾丸和附睾。睾丸切片的组织学观察表明，Slx 2基因敲除不影响精子发生的三个主要阶段：有丝分裂，减数分裂和精子发生。此外，我们通过免疫荧光分析进一步证实了Slx 2的破坏不影响精原干细胞的数量、减数分裂进程或XY体的形成。由于Slx 2基因敲除小鼠的精子发生正常，因此这些小鼠具有生育能力。综上所述，我们表明Slx 2本身不是小鼠精子发生的必需基因，CRISPR/Cas9技术可以加快睾丸特异性X连锁基因的体内功能研究。
Mammalian spermatogenesis is a highly regulated multistage process of sperm generation. It is hard to uncover the real function of a testis specific gene in vitro since the in vitro model is not yet mature. With the development of the CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated 9) system, we can now rapidly generate knockout mouse models of testis specific genes to study the process of spermatogenesis in vivo. SYCP3-like X-linked 2 (SLX2) is a germ cell specific component, which contains a Cor1 domain and belongs to the XLR (X-linked, lymphocyte regulated) family. Previous studies suggested that SLX2 might play an important role in mouse spermatogenesis based on its subcellular localization and interacting proteins. However, the function of SLX2 in vivo is still elusive. Here, to investigate the functions of SLX2 in spermatogenesis, we disrupted the Slx2 gene by using the CRISPR/Cas9 system. Since Slx2 is a testis specific X-linked gene, we obtained knockout male mice in the first generation and accelerated the study process. Compared with wild-type mice, Slx2 knockout mice have normal testis and epididymis. Histological observation of testes sections showed that Slx2 knockout affected none of the three main stages of spermatogenesis: mitosis, meiosis and spermiogenesis. In addition, we further confirmed that disruption of Slx2 did not affect the number of spermatogonial stem cells, meiosis progression or XY body formation by immunofluorescence analysis. As spermatogenesis was normal in Slx2 knockout mice, these mice were fertile. Taken together, we showed that Slx2 itself is not an essential gene for mouse spermatogenesis and CRISPR/Cas9 technique could speed up the functional study of testis specific X-linked gene in vivo.