Generation Of Autosomal Dominant Polycystic Kidney Disease Model In Mini-pig

Autosomal dominant polycystic kidney disease (ADPKD) is a common dominant renal disorder caused by mutations in either PKD1or PKD2, with an incidence of1/400～1/1000. Its key feature is fluid-filled cysts in bilateral kidneys, and as the illness deteriorates, both number and volume of cysts increase. Approximately half of ADPKD patients develop end stage renal disease (ESRD) at their fifth or sixth decade of life. Until now, there is no effective therapy for ADPKD except for renal dialysis or kidney transplantation. The research in mouse models of ADPKD has promoted people’s understanding about the pathogenesis of ADPKD to a large extent; however, mouse models have not yet brought about an effective therapy. Contrasted to the obvious disparity between mouse and people in kidney, the mini-pig owns more similarities with human in kidney, which makes the mini-pig an excellent model to study renal diseases. Herein, I try to establish the ADPKD mini-pig model in this study, so that it will serve as a novel model faithfully recapitulating human ADPKD, and promote the study of etiology as well as development of effective therapeutic interventions.The SBM mice represent a transgenic model of ADPKD by overexpressing c-Myc in kidneys. Therefore, c-Myc was overexpressed in pig’s kidneys in an effort to imitate the SBM mice. The pKsp-c-Myc-b transgene vector was constructed using mouse Ksp-cadherin promoter and the cDNA of pig’s c-Myc gene. After somatic cell nuclear transfer (SCNT), seven c-Myc transgenic piglets were delivered. Three transgenic piglets died soon after birth, displaying no obvious abnormality in kidneys at necropsy. Western Blot analysis showed that c-Myc was overexpressed in the brain, heart, liver and kidney of c-Myc transgenic piglet (No.3). Immunohistochemical staining of kidney sections revealed high level of c-Myc within the epithelial cells of renal tubules in piglet No.3. However, except for slight dilation of renal tubules, no cysts were observed. When c-Myc transgenic pigs were1,4,6months old, blood sample were collected to measure blood urea nitrogen (BUN) and serum creatinine (Scr). At1month, c-Myc transgenic pigs had significantly higher BUN level than the wild-type littermates, but this difference did not persist to4months or6months. No difference in Scr was detected between transgenic pigs and wild-type pigs at all three time points. When c-Myc transgenic pig No.10turned13months old, kidney sample was collected. Western Blot results revealed that there was no difference in the protein level of c-Myc, total ERK1/2and phosphorylated ERK1/2between transgenic pig No.10and a wild-type pig. No noticeable renal abnormalities were found through H&E staining of kidney sections from transgenic pig No.10. Therefore, c-Myc transgenic pigs did not develop ADPKD, implying the uniqueness of SBM mice.In order to introduce a missense mutation (hypomorphic allele) into pig PKD2gene, seven pairs of TALEN targeting the9th exon of pig PKD2gene were screened for changing the658th amino acid of polycystin-2from Leu to Trp. Both sequencing and EcoR V digestion analysis demonstrated that T-93, T-931and T-934were able to cause mutations in target sites, while T-93had the highest efficiency. Upon37℃cell culture condition, T-93yielded mutation frequency of6%～7%, and if cells were applied to transient hypothermia (30℃) after nucleofection, the mutation frequency rose to15%～17%. T-93, T-931and T-934had almost the same target site, except that the binding sites of T-931and T-934was several base pairs longer than those of T-93. The extended binding sites led to decreased mutation frequency. Once T-93and single-stranded DNA (ssDNA) donor template were co-transfected into Chinese experimental mini-pig fetal fibroblasts, the cell viability dropped dramatically, so further optimization was needed to improve cell condition.Moreover, CRISPR-Cas9technique was exploited to knock out the pig PKD2gene. Within all11target sites on different exons of PKD2,6targets sites were mutated, while pX330-1had the highest mutation rate of11%. Because the target site of pX330-1was on the first exon, it was expected to end in complete inactivation of polycystin-2.As reported in human, mouse and rabbit, CDH16is specifically expressed in the kidney. A kidney-specific promoter is needed to knock out PKD2gene in a kidney specific manner. Therefore, the pig CDH16gene was identified in this study. The pig CDH16gene had18exons, and its transcripts were detected in both the kidney and lung of Chinese experimental mini-pig, with relatively higher transcription level in the kidney. Dual-luciferase assay proved that the pig CDH16promoter was able to drive transcription in LLC-PK1cells.In summary, pKsp-c-Myc-b transgenic mini-pig did not develop ADPKD. Screened TALEN and CRISPR-Cas9could be used to introduce hypomorphic PKD2allele or knock out PKD2gene in mini-pig. At the same time, the identified pig CDH16promoter could be utilized to re-construct current CRIPSR-Cas9vector and constrain Cas9expression mainly in the kidney, thus knocking out pig PKD2gene in a kidney specific manner.