Establishment Of The Kidney Deficient Pig Model Via Knocking Out The Six1 And Six4 Genes

Background:The intra-and interspecies blastocyst complementation,which has been demonstrated using rodent models and large mammals,raises an intriguing possibility to generate human organs in easy accessible host animals.In order to generate patient-specifc organs,host animals lacking the ability to generate various organs would be required.Considering its resemblance to humans in anatomy,physiology,organ size,genomic similarity and cell cycle characterisics,pig consitutes a good candidate for such purpose.Kidney development is a continuous and complex process that requires constant regulation of multiple genes.The mammalian Six gene family consists of six members(Six1-6),of which Six1 and Six4 are expressed in the nephrogenic mesenchyme and play an critical roles in kidney development.A lack of Six1 in mice leads to uni-or bilateral renal hypoplasia and a failure of ureteric bud to form branching morphogenesis.Knockout of the Six1 and Six4 genes can result in severe developmental defects in the ureteric bud and the metanephric mesenchyme,and ureters and bilateral kidneys failed to develop in all the Six1/Six4-deficient mice.However,it remains unknown whether the function of Six1 and Six4 are conserved across distant species and the disruption one or both of Six1 and Six4 genes can disable kidney development in pigs.ObjectiveIn this study,we attempt to generate the Six1-/-and Six1-/-Six4-/-porcine fetuses via CRISPR/Cas9 system combined with SCNT technology.The functions of Six1 and Six4 genes on porcine fetal tissue and organ development,especially kidney development were investigated.We also attempt to investigate the effects of knockout of the Six1 and Six4 genes on kidney development in pigs and whether kidney-deficient pig models can be obtained.MethodsThe first exon of Six1 and Six4 gene was chosen for the target to design the sgRNA and Cas9/sgRNA of the Six1 gene and Six4 gene were respectively constructed.The efficient Cas9/sgRNA was co-transfected with the G418 resistant plasmid(pCMV-tdTomato)into the primary porcine fetal fibroblasts(PFFs).The monoclonal cell populations were obtained through the G418 screening and the genotypes of monoclonal cells were identified by sequencing analysis.The biallelic knockout cell colonies were chosen as donor cell for SCNT to generate the Six1-/-and Six1-/-Six4-/-porcine fetuses.Genotyping of pig fetuses was confirmed via sequencing;Western Blot and immunohistochemistry were used to further determine whether Six1 and Six4 proteins were expressed in knockout fetuses;The kidney phenotypes were identified by dissection,HE staining,Western Blot,and immunohistochemistry.ResultsCas9/sgRNA expression vectors for Six1 and Six4 gene targeting were constructed successfully.Total 48 monoclonal cell populations with presumed Six1 gene mutation were obtained and identified by PCR,among them 21 cell colonies showed Six1 bialellic mutation(Six1-/-).By the same way,we obtained total 44 monoclonal cell populations for both Six1 and Six4 targeting and 13 of them showed Six1-/-Six4-/-genotypes.Genotype analysis showed that the three fetuses were all biallelic Six1 gene mutants and 16 fetuses were all Six1/Six4 knockout mutants,corresponding to the donor cells respectively.Six1 and Six4 proteins had not been detected in the Six1 and Six4 mutant fetuses.Further studies showed that the knockout of the Six1 gene caused renal hypoplasia in pigs,and the ureteral buds were unable to branch to form a complete collecting duct system.Both Six1 and Six4 gene knockout caused a failure of kidney development in pigs.ConclusionIn this study,we produced the Six1-/-and Six1-/-/Six4-/-porcine fetuses via CRISPR/Cas9 system combined with SCNT technology,which provide a powerful platform for the studies of the mechanisms of kidney development and the organogenesis via the blastocyst complementation.