Testis Mediated ZFN Targeting TMEM18in Mouse Germ Cells

Using zinc finger nucleases (ZFN) for gene knockout is very simple and efficient, and has become a hot research field in recent years. Although knockout gene using ZFN seems simple and efficient, however, the technology in the present successful instances still needs complex operation on embryo and microinjection, which are difficult for general laboratory. To solve this problem, we intend to establish a more convenient method of gene knockout technology using ZFN, which is referred as testis-mediated zinc finger nucleases (TM-ZFN) gene knockout technology. Transmembrane protein18(TMEM18,) is associated with obesity, but its function is still unknown. We want to construct a ZFN for targeting TMEM18, and to explore its functions.A universal plasmid, named p2NfokIT, was constructed for establishment of zinc finger nucleases. This plasmid contains two components which are nuclear localization peptide linked to endonuclease fok Ⅰ(NLS-fokI). These two components were connected by T2A. The two NLS-fokIs, respectively, contained two restriction sites. Just to insert DNA binding domain of a zinc finger protein into the plasmid, a plasmid for expressing zinc finger nucleases will be easily constructed.In order to establish a ZFN for targeting TMEM18, the sequence of TMEM18was scanned. A more ideal targeting site of ZFN in its third exon was found. The zinc finger and two DNA binding domains of zinc finger protein to identify this site were designed. One of DNA binding domains of zinc finger protein was constructed into SpIC skeleton, the other into Zif268skeleton. These DNA binding domains of zinc finger proteins was respectively synthesized by fusion PCR using six primers, and inserted into the universal plasmid p2NfokIT. A plasmid p2NfokIT/2ZF expressing zinc finger nucleases for targeting TMEM18was successfully constructed.The activity of artificial zinc nuclease was assessed using a reporter gene assay. In this assay, an eGFP-mutation plasmid pMeGFP, containing eGFP with a stop codon and TMEM18targeting sites, and an eGFP donor plasmid without start codon and upstream promoter in an eGFP were constructed. pMeGFP, pddeGFP and p2NfokIT/2ZF were co-transfected into HEK293cells. After48hours, the fluorescence of the cells was detected. The results showed that ZFN can effectively target expected site.The analysis of protein level showed that TMEM18was highly expressed in mouse spermatogenic cells, spermatocytes and mature sperm.100ul of liposome mixture containing10μg of ZFN plasmid and Lipfectimine2000was respectively injected into two testes of one mouse (50ul each). Three days later, protein levels of germ cells in the testes and mature sperms in epididymis were analyzed. The results showed that protein levels of TMEM18in mature sperms was significantly reduced, but not significantly changed in testis. The results of the PCR amplification of the targeting site and the analysis of the PCR products by enzyme digestion showed that ZFN could effectively cut down the targeting sites of the mature sperm genomic DNA. These results suggest that testicular injection of ZFN is an efficient gene knock method, and testis-mediated ZFN to create knockout mice is feasible.Our research was suggested that knocking out TMEM18on Embryo may influence the implantation of fertilized eggs. The protein levels of β-catenin and phosphorylated Akt, which are related to fertilized egg implantation, were detected in the primary mouse skeletal muscle cells transfected with ZFN plasmid. The results showed that the protein level of TMEM18, β-catenin and phosphorylated Akt were significantly decreased, indicating that TMEM18knocking out, resulting in damages to the Wnt signaling pathway and insulin signaling pathway. These damages may affect implantations of the fertilized eggs. These results provides a new direction for us to continue the studies of the function of TMEM18.