Generation Of Tryptophan Hydroxylase Gene Knockout Pigs By CRISPR/Cas9-mediated Gene Targeting

Background:Tryptophan hydroxylase 2(TPH2)is the limiting velocity enzymes in the biosynthesis of the hormone and neurotransmitter serotonin(5-HT)in nerve cell of central nervous system.It involves in stress adjustment along with hypothalamic-pituitary-adrenal axis(HPA)and sympathetic nervous system.Stress is the key to the question of the pathogenesis of emotional disorders which causes the clinical common mental disease such as anxiety disorder and depressive disorder.While the extensive use of Tph2-/-mouse had made great progress in understanding various neurobehavior processed related to 5-HT deficiency,there is a huge difference between humans and mice in terms of physiology and gene expression.Pigs are considered as an ideal large animal model in biomedical research because they share many similarities with humans in trems of anatomy and physiology.In this study,highly efficient CRISPR/Cas9 system will be used to target and disrupt Tph2 gene in Bama miniature pigs followed by somatic cell nuclear transfer to produce the Tph2-/-pigs.These Tph2-/-pigs could be useful models for providing further insight into pathogenesis of behavior disorders.Objective:To generate Tph2-/-Bama miniature pigs model by using the CRISPR/Cas9 to knockout the Tph2 gene of Bama miniature pigs.These Tph2-/-pigs will be useful for studies on the neurobehavior regulated by serotonergic neurotransmission.Methods:Targeting sgRNA 1 and sgRNA 2 for the Tph2 coding region were designed using online tools(http://crispr.mit.edu/)and synthesized by Genscript Co.,Ltd.(Nanjing,China).Oligos of sgRNAs were annealed and sub-cloned into BbsI-digested pX330 plasmid,respectively.Approximately 1×106 PFFs were transfected with 1 p.g Tph2 targeting Cas9 plasmid and 0.2 ?g neomycin-expression plasmid(pCMV-tdTomato).G418 was used to screen the postitive colonies.The PCR products of each colony were sequenced andbiallelic modified Tph2 coloines were identified.Then,Tph2-/-pigs were produced by SCNT technology and the genotpyes were confirmed by PCR sequencing using genomic DNA isolated from their punched ear tissue.Results:Targeting sgRNA 1 and sgRNA 2 for the Tph2 coding region were designed using online tools(http://crispr.mit.edu/).Their cleavage efficiencies were compared and sgRNAl was chosen for further Tph2 gene targeting experiment.The results indicated that 12 colonies had monoallelic modifications in the Tph2 targeting region and 20 had biallelic modifications.About 38.5%of the cell colonies had biallelic mutations.To produce Tph2-/-piglets,PFFs colonies with biallelic mutations were mixed as donor cells for SCNT.Totally,12 live-born piglets without displaying any obvious developmental defects or differences from wild-type neonates have been born.To determine the TPH2 protein and 5-HT expression level of the gene targeted piglets,Western blot and immunohistochemistry analysis were carried out.Conclusion:CRISPR/Cas9 combined with SCNT to produce Tph2-/-piglets without displaying any obvious developmental defects or differences from wild-type neonates.Our results showed that CRISPR/Cas9 technology is an efficient tool to produce genetically modified pigs which are suitable for study 5-HT system function.