Kidney Development Of Pig Fetus And Construction Of Pig Cell Line With Pax2 Knocked-out

BackgroundAs the number of patients with end-stage renal disease continues to increase,the shortage of donors for kidney transplant has received widespread attention from researchers.The use of induced pluripotent stem cells（iPSC）and blastocyst complementation to recreate humanized kidneys in animal body is expected to be a new source of donors for human kidney transplants.The prerequisite for reconstituting humanized kidney by this method is to obtain an animal model of renal agenesis.Pigs are considered to be ideal animals for providing niche for human iPSC development and differentiation because they are readily available and their phenotype in many cases is more similar to humans,e.g.anatomy,physiological function and organ size.Paired box 2（Pax2）is an important transcription factor in mammalian embryonic kidney development.It is widely expressed in pronephros,mesonephros and metanephros,and interacts with various factors regulating kidney development,including accurate induction of nephrogenic cord formation,formation and differentiation of nephric duct,growth and branching of ureteric buds,and differentiation of metanephric mesenchyme.Pax2 mutations display renal defects in mice.However,the development of kidneys and the expression of related signaling molecules are species specific in time and space.Therefore,investigating the development of pig kidneys and the expression of Pax2and other signaling molecules during kidney development,and constructing Pax2knockout pig cell line lays the experimental foundation for the subsequent construction of a pig model with kidney defects or agenesis,and also provides a theoretical basis for the future regeneration of humanized kidney in pigs.ObjectiveTo clarify the developmental status of porcine fetal kidney at different stages,and to study the expression of signaling molecules related to the kidney development.To construct Pax2 knockout pig cell lines using CRISPR/Cas9（Clustered regularly interspaced short palindromic repeat/CRISPR-associated system）gene editing technology.MethodsThe kidney tissues of porcine fetuses at different stages（fetuses on post-implantation days 21.5,27.5,35,45,75 and 110）were collected.The histological structures of porcine fetal kidneys were observed by hematoxylin-eosin（H&E）staining.The mRNA and protein expression levels of factors related to renal development signaling molecules（Pax2,Pax8,Six2,Six1,Sall1 and Bmp4）were detected by quantitative real-time PCR（qPCR）and Western blot,respectively.The expression and localization of Pax2 at different stages of porcine fetal kidney were detected by immunohistochemistry.The CRISPR/Cas9 gene editing technology was used to design the sgRNA of the Pax2 gene of Ba-Ma mini pig and construct the target plasmid.The target plasmid and pCMV-tdTomato plasmid（with the G418 resistance）were co-transfected 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 cell lines,in which the Pax2 gene was successfully knocked out,were finally determined.ResultsOn the 27.5^th day,the metanephros of the porcine fetuses has begun to develop;on the 35^th day,the early renal corpuscle has appeared,and the renal tubules began to develop;on the 45^th day,the immature glomeruli in the cortex region and the proximal matured renal corpuscles in the medulla region were observed.The number of nephrons on the 75^th day increased significantly,and kidney pyramid,renal papillae and renal pelvis were observed at this stage.On the 110^th day,kidney was basically mature,the cortex and medulla could be distinguished clearly,and new nephrons kept to be produced.The mRNA and protein expression levels of Pax2,Pax8,Six2,Six1,Sall1and Bmp4 on 75d were higher than that on 35d.The expression localization of Pax2protein at 27.5d,35d,45d,75d and 110d was further analyzed by immunohistochemical.The results showed that Pax2 was widely expressed in ureteric bud,metanephric mesenchyme,cap mesenchyme,renal vesicles,comma--shaped bodies,S-shaped bodies and renal tubule,and is continuously expressed during the development of the metanephros.The sgRNA of Pax2 gene of Ba-Ma mini pig was designed and the CRISPR/Cas9targeting vector was constructed.After transfecting primary porcine fetal fibroblasts,73 monoclonal cells were obtained after G418 screening.After identification,40 cell clones with Pax2 mutation were obtained,and the mutation efficiency reached 54.8%.33 cell clones of them were Pax2 biallelic mutant and these cell clones could be used for the next somatic cell nuclear transfer experiment.The biallelic knockout efficiency was 45.2%.Therefore,the Pax2 knockout pig cell line were successfully constructed,which laid the experimental foundation for the production of the Pax2 knockout pig model.