Abstract
To combat organ shortage in transplantation medicine, a novel strategy has been proposed to generate human organs from exogenous pluripotent stem cells utilizing the developmental mechanisms of pig embryos/foetuses. Genetically modified pigs missing specific organs are key elements in this strategy. In this study, we demonstrate the feasibility of using a genome-editing approach to generate anephrogenic foetuses in a genetically engineered pig model. SALL1 knockout (KO) was successfully induced by injecting genome-editing molecules into the cytoplasm of pig zygotes, which generated the anephrogenic phenotype. Extinguished SALL1 expression and marked dysgenesis of nephron structures were observed in the rudimentary kidney tissue of SALL1-KO foetuses. Biallelic KO mutations of the target gene induced nephrogenic defects; however, biallelic mutations involving small in-frame deletions did not induce the anephrogenic phenotype. Through production of F1 progeny from mutant founder pigs, we identified mutations that could reliably induce the anephrogenic phenotype and hence established a line of fertile SALL1-mutant pigs. Our study lays important technical groundwork for the realization of human kidney regeneration through the use of an empty developmental niche in pig foetuses.
Highlights
To combat organ shortage in transplantation medicine, a novel strategy has been proposed to generate human organs from exogenous pluripotent stem cells utilizing the developmental mechanisms of pig embryos/foetuses
We report here the results obtained after introducing SALL1 mutations in pigs, including the mutation efficiencies after cytoplasmic injection of transcription activator-like effector nucleases (TALENs) and CRISPR/Cas[9] in zygotes, the effects of the mutations on kidney formation in foetuses and progeny, and our success in establishing a line of SALL1-mutant pigs characterized by an anephrogenic phenotype
Mutations were induced in over 80% of blastocysts obtained after injection of 2 or 5 ng/μl Platinum TALEN-encoding mRNA (Table 1)
Summary
To combat organ shortage in transplantation medicine, a novel strategy has been proposed to generate human organs from exogenous pluripotent stem cells utilizing the developmental mechanisms of pig embryos/foetuses. Generating transplantable solid organs in vitro is considered impractical owing to the complex three-dimensional structure and function of human organs[4,5] Under such circumstances, the novel concept of growing human organs in vivo by utilizing the developmental mechanisms of porcine embryos genetically engineered to be unable to form a specific organ has attracted considerable attention [for review, see6,7]. Kobayashi et al.[16] and Usui et al.[17] successfully produced xenogeneic pancreases in Pdx[1] KO mice and allogeneic kidneys in Sall[1] KO mice, respectively, using a blastocyst complementation approach We report here the results obtained after introducing SALL1 mutations in pigs, including the mutation efficiencies after cytoplasmic injection of transcription activator-like effector nucleases (TALENs) and CRISPR/Cas[9] in zygotes, the effects of the mutations on kidney formation in foetuses and progeny, and our success in establishing a line of SALL1-mutant pigs characterized by an anephrogenic phenotype
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