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. 2022 Aug 2;11(15):2378. doi: 10.3390/cells11152378

Figure 7.

Figure 7

Production of double-transgenic hNGF-TG/pNGF-KO pigs. (A) The endogenous pNGF gene locus. (B) The map of the donor plasmid pLA-hNGF-RFP-RA used for replacement of the endogenous pNGF gene with the foreign hNGF gene by CRISPR/Cas9 system combined with homologous recombination (HR). LA and RA: left and right homologous arms, respectively. (C) The endogenous pNGF gene locus after HR. P1-F and P1-R are a set of primers used for PCR identification of clones with its endogenous pNGF gene being replaced by the foreign hNGF gene. (D) RFP-expressing pig fibroblast cell colonies resulted from selection of the no. 5 F0 hNGF-TG pig-derived fibroblasts co-transfected with the donor plasmid pLA-hNGF-RFP-RA, and the CRISPR/Cas9 system expression plasmids (ppNGF-sgRNA5-Cas9 and ppNGF-sgRNA10-Cas9) that target the pNGF gene. (E) PCR identification of selected RFP-expressing fibroblast cell colonies with their endogenous pNGF gene being replaced by the foreign hNGF gene. He: heterozygous cell colonies. FP: false positive. Ho: homozygous cell colonies, which were used as donor cells for subsequent SCNT. (F) EGFP and RFP expression in double-transgenic hNGF-TG/pNGF-KO pigs generated by SCNT. St: stomach. He: heart. To: tongue. Pa: parotid gland. (G) PCR identification of hNGF-TG/pNGF-KO pigs with their endogenous pNGF gene being replaced by the foreign hNGF gene. WT: wild-type pig. 31, 32, 33, and 34 are the ID no. of four hNGF-TG/pNGF-KO pigs generated by SCNT. (H) The sequencing result of the PCR amplification product of the endogenous pNGF gene locus in hNGF-TG/pNGF-KO pigs. (I) Average hNGF and pNGF protein concentration in different tissues of hNGF-TG/pNGF-KO pigs (no. 31 and no. 32) died within one day after birth. No pNGF was detected in examined tissues of hNGF-TG/pNGF-KO pigs. Sm: submandibular gland. Sl: sublingual gland. Pa: parotid gland. He: heart. Fa: fat. Lu: lung. Mu: muscle. (J) Average hNGF and pNGF concentration in oral saliva collected at rest time from survived hNGF-TG/pNGF-KO pigs (no. 33 and no. 34) at the age of 5 days. No pNGF was detected in oral saliva of hNGF-TG/pNGF-KO pigs. ** means the difference between two groups reached statistical significance at p < 0.01.