Fig. 3. The preparation of hIAPP miniature pigs.

a Experimental flow of IAPP-humanized pig construction. ① Isolation and culture of miniature PFFs; ② Transfection of miniature PFFs; ③ Inoculate cells into 10 cm culture dishes using the limiting dilution; ④ Monoclonal cell picking; ⑤ Repeat step ④; ⑥ Positive colonies were screened by restriction enzyme digestion assay; ⑦ Expansion of culture-positive clones; ⑧ Somatic cell nuclear transfer; ⑨ Embryo transfer; ⑩ Identification of hIAPP cloned pigs. b, c Positive cloned cells were identified by restriction enzyme digestion assay and verified by Sanger sequencing. d Statistics of blastocyst development were performed on 4 positive clones. 8.5 days after nuclear transfer, the cells that could develop normally into blastocysts, and the results of nuclear staining also prove this conclusion. e Statistical analysis of the blastocyst development rate of positive cloned cells. No significant differences were found compared to the control group