Laboratory of Animal Reproduction, Faculty of Bioscience and Bioindustry, Tokushima University, Tokushima, Tokushima, Japan.
Mol Reprod Dev. 2020 Apr;87(4):471-481. doi: 10.1002/mrd.23335. Epub 2020 Mar 12.
Pancreatic duodenal homeobox 1 (PDX1) is a crucial gene for pancreas development during the fetal period. PDX1-modified pigs have the potential to be used as a model of diabetes mellitus. However, the severe health problems caused by the PDX1 mutation limit phenotypic studies of PDX1-modified pigs as diabetes models. In this study, we generated PDX1-modified pigs by the CRISPR/Cas9 system introduced into zygotes via electroporation and investigated the mosaicism, phenotypes, and inheritance of the resulting pigs. After the embryo transfer of PDX1-modified zygotes, nine mutant piglets were delivered. Two piglets were apancreatic biallelic mutants. For the other seven piglets, the ratio of mutant alleles to total alleles was 17.5-79.7%. Two mutant piglets with high mutation rates (67.7% and 79.7%) exhibited hypoplasia of the pancreas, whereas the other five piglets were healthy. One of the male mutant piglets was further analyzed. The ejaculated semen from the pig contained PDX1-mutant spermatozoa and the pig showed normal reproductive ability. In conclusion, the frequency of the PDX1 mutation is presumed to relate to pancreas formation, and PDX1 mutant founder pigs generated from zygotes introduced to the CRISPR/Cas9 system can serve as providers of nonmosaics to contribute to medical research on diabetes mellitus.
胰腺十二指肠同源盒 1（PDX1）是胎儿期胰腺发育过程中的关键基因。PDX1 修饰猪有可能被用作糖尿病模型。然而，PDX1 突变引起的严重健康问题限制了 PDX1 修饰猪作为糖尿病模型的表型研究。在这项研究中，我们通过电穿孔将 CRISPR/Cas9 系统引入受精卵，生成了 PDX1 修饰猪，并研究了所得猪的嵌合体、表型和遗传。在 PDX1 修饰胚胎移植后，产下了 9 头突变猪。其中 2 头是胰腺双等位基因突变纯合子。对于其他 7 头猪，突变等位基因与总等位基因的比例为 17.5-79.7%。2 头突变率较高（67.7%和 79.7%）的突变猪表现为胰腺发育不良，而其他 5 头猪则健康。对其中一头雄性突变猪进行了进一步分析。该猪的射出精液中含有 PDX1 突变精子，且具有正常的生殖能力。总之，PDX1 突变的频率似乎与胰腺形成有关，从引入 CRISPR/Cas9 系统的受精卵中生成的 PDX1 突变奠基猪可以作为非嵌合体的提供者，为糖尿病的医学研究做出贡献。
