CRISPR/Cas9 Promotes Functional Study of Testis Specific X-Linked Gene In Vivo

Li M, Huang R, Jiang X, Chen Y, Zhang Z, Zhang X, Liang P, Zhan S, Cao S, Songyang Z, Huang J.
Source: PLoS ONE
Publication Date: (2015)
Issue: : e0143148
Research Area:
Gene Expression
Molecular Biology
Cells used in publication:
Embryonic Stem Cell (ES), human
Species: human
Tissue Origin: embryo
4D-Nucleofectorâ„¢ X-Unit
V6.5 mouse embryonic stem (ES) cells were cultured with standard ES cell culture conditions. The gRNA ligated pX330 vectors were transfected into V6.5 ES cells using P3 primary cell 4D-nucleofecton kit (Lonza, V4XP-3024) according to the manufacturer’s instructions.
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.