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. 2024 Sep 11:26:783-791.
doi: 10.1016/j.reth.2024.08.025. eCollection 2024 Jun.

Generation of insulin-like growth factor 1 receptor-knockout pigs as a potential system for interspecies organogenesis

Masaki Nagaya  1 , Ayuko Uchikura  1 , Kazuaki Nakano  1   2 , Masahito Watanabe  1   2 , Hitomi Matsunari  1   2 , Kazuhiro Umeyama  1   2 , Naoaki Mizuno  3   4 , Toshiya Nishimura  5 , Hiromitsu Nakauchi  3   4   5 , Hiroshi Nagashima  1   6   2
Affiliations

Generation of insulin-like growth factor 1 receptor-knockout pigs as a potential system for interspecies organogenesis

Masaki Nagaya et al. Regen Ther. .

Abstract

Background: To overcome organ shortage during transplantation, interspecies organ generation via blastocyst complementation has been proposed, although not yet in evolutionarily distant species. To establish high levels of chimerism, low chimerism is required early in development, followed by high chimerism, to effectively complement the organ niche. Very few human cells are expected to contribute to chimerism in heterologous animals. Previous studies had demonstrated increased donor chimerism in both intra- and interspecies chimeras in rodents, using insulin-like growth factor 1 receptor (Igf1r) knockout (KO) mice; deletion of the Igf1r gene in the mouse host embryo created a cell-competitive niche. The current study aimed to generate IGF1R-KO pigs and evaluate whether they have the same phenotype as Igf1r-KO mice.

Methods: To generate IGF1R-KO pigs, genome-editing molecules were injected into the cytoplasm of pig zygotes. The fetuses were evaluated at 104 days of gestation.

Results: IGF1R-KO pigs were generated successfully. Their phenotypes were almost identical to those of Igf1r-KO mice, including small lungs and enlarged endodermal organs in fetuses, and they were highly reproducible.

Conclusions: Pigs may allow the generation of organs using blastocyst complementation with developmentally-compatible xenogeneic pluripotent stem cells over a large evolutionary distance.

Keywords: Blastocyst complementation techniques; Growth retardation; Insulin-like growth factor 1 receptor; Pig.

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Conflict of interest statement

H.Nagashima is a founder and shareholder of PorMedTec Co., Ltd. These associations do not alter the authors' adherence to the journal's policies on sharing data and materials. The other authors declare that they have no conflicts of interest.

Figures

Fig. 1
Fig. 1
Generation of IGF1R-gene-knockout porcine fetuses by genome editing. Schematic structure of the porcine IGF1R gene, which consists of 21 exons, and the design of CRISPR-Cas9 targeting the IGF1R gene. The coding regions are indicated by black vertical lines. Two different gRNAs (gRNA1 and gRNA4) designed in exons 2 and 9 and used for the generation of IGF1R-gene-knockout fetuses are shown. The gRNAs and protospacer adjacent motif (PAM) sequences are underlined and boxed, respectively.
Fig. 2
Fig. 2
Evaluation of each IGF1R-targeting guide RNA. (A) Guide RNA (gRNA) of various concentrations was used in nuclease based-mutation detection assays to determine the genome targeting efficiency. Genomic DNA was extracted from fibroblasts for IGF1R-locus analysis tracking indels by decomposition. Subsequently, the efficiency of mutation induction by cytoplasmic injection of IGF1R-targeted CRISPR-Cas9 into parthenogenetic embryos was evaluated. N: non-digest, d: nuclease-digest, Indel: insertion/deletion. (B) Blastocyst formation was compared for various concentrations of each guide RNA (Exon 2: gRNA; 5 ng/μl and Exon 9: gRNA; 2–20 ng/μl). Blastocyst formation rates for each of exons 2 and 9 were reliable (exon 2: 41.7–42.1%, exon 9: 50.7–64.4%). Scale: 500 μm.
Fig. 3
Fig. 3
Appearance of IGF1R–KO porcine fetuses. IGF1R–KO fetuses were obtained by anatomical dissection at 104 d of fetal age. The phenotype differs from that of IGF1R–KO fetuses not only in the size of the bodies, thymus, and lungs, but also in the appearance of the skin, which is markedly opaque. The translucent skin of the IGF1R–KO fetuses was notable. K363-7 (KO female. exon 2: gRNA1) and K363-6 (WT female) were obtained from the same litter. K366: 2–5 (KO. exon 9: gRNA4). KO: knockout, WT: wild type. Scale bars: white = 1 cm; black = 2 cm.
Fig. 4
Fig. 4
Body and organ weight ratios of IGF1R–KO porcine fetuses. Wet organ weight was determined by anatomic dissection at 104 d of fetal age. (A) Body weight and organ weight. The three graphs compare KO and WT, and male and female KO porcine fetuses. (B) Each organ is represented as organ per unit body weight. Quantitative data are presented as means ± standard error of mean. KO: knockout, WT: wild type. IGF1R–KO fetuses: n = 5 (3 females and 2 males), WT fetuses: n = 9; ∗P < 0.05 versus WT.
Fig. 5
Fig. 5
Histological appearance of organs of IGF1R–KO porcine fetuses obtained by anatomical dissection at 104 d of fetal age. (A–C) Hematoxylin and eosin (H&E) staining (A), IGF1R staining (B), and Western blot analysis (C) are shown. (A) H&E staining of each organ of IGF1R–KO porcine fetuses at 104 d of age. There was no apparent difference between KO and WT fetuses in any organ. (B) Immunohistochemical staining of IGF1 in each organ of IGF1R–KO porcine fetuses at 104 d of age. IGF1R staining was negative in each organ of the KO fetuses and positive in the WT fetuses. KO involved a K366-3 female and a K366-2 male. WT was a K366-6 female. KO: knockout, WT: wild type. Scale bar: 100 μm. (C) Western blotting was negative for all organs tested in KO fetuses. Lu: lungs, H: heart, P: pancreas, T: thymus, K: kidneys, Li: liver. KO: knockout, WT: wild type.
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