Zhu Xiangxing, Wei Yanyan, Zhan Qunmei, Yan Aifen, Feng Juan, Liu Lian, Tang Dongsheng
Guangdong Provincial Engineering and Technology Research Center for Gene Editing, School of Medical Engineering, Foshan University, Foshan 528225, China.
Guangdong Provincial Key Laboratory of Animal Molecular Design and Precise Breeding, School of Life Science and Engineering, Foshan University, Foshan 528225, China.
Animals (Basel). 2020 Mar 17;10(3):501. doi: 10.3390/ani10030501.
Bama minipigs are a local pig breed that is unique to China and has a high development and utilization value. However, its high fat content, low feed utilization rate, and slow growth rate have limited its popularity and utilization. Compared with the long breeding cycle and high cost of traditional genetic breeding of pigs, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) endonuclease 9 system (CRISPR/Cas9)-mediated gene editing can cost-effectively implement targeted mutations in animal genomes, thereby providing a powerful tool for rapid improvement of the economic traits of Bama minipigs. The iroquois homeobox 3 () gene has been implicated in human obesity. Mouse experiments have shown that knocking out significantly enhances basal metabolism, reduces fat content, and controls body mass and composition. This study aimed to knock out using the CRISPR/Cas9 gene editing method to breed Bama minipigs with significantly reduced fat content. First, the CRISPR/Cas9 gene editing method was used to efficiently obtain cells. Then, the gene-edited cells were used as donor cells to produce surviving Bama minipigs using somatic cell cloning. The results show that the use of cells as donor cells for the production of somatic cell-cloned pigs results in a significant decrease in the average live litter size and a significant increase in the average number of stillbirths. Moreover, the birth weight of surviving somatic cell-cloned pigs is significantly lower, and viability is poor such that all piglets die shortly after birth. Therefore, the preliminary results of this study suggest that IRX3 may have important biological functions in pigs, and should not be used as a gene editing target to reduce fat content in Bama minipigs. Moreover, this study shows that knocking out does not favor the survival of pigs, and whether targeted regulation of in the treatment of human obesity will also induce severe adverse consequences requires further investigation.
巴马香猪是中国特有的地方猪种，具有较高的开发利用价值。然而，其脂肪含量高、饲料利用率低、生长速度慢，限制了其推广和利用。与传统猪遗传育种周期长、成本高相比，成簇规律间隔短回文重复序列（CRISPR）/CRISPR相关蛋白9（Cas）核酸酶9系统（CRISPR/Cas9）介导的基因编辑能够经济高效地在动物基因组中实现靶向突变，从而为快速改良巴马香猪的经济性状提供了有力工具。Iroquois同源盒3（IRX3）基因与人类肥胖有关。小鼠实验表明，敲除IRX3可显著提高基础代谢率、降低脂肪含量并控制体重和身体组成。本研究旨在利用CRISPR/Cas9基因编辑方法敲除IRX3，培育脂肪含量显著降低的巴马香猪。首先，利用CRISPR/Cas9基因编辑方法高效获得IRX3基因编辑细胞。然后，将基因编辑细胞作为供体细胞，通过体细胞克隆生产存活的IRX3基因编辑巴马香猪。结果表明，使用IRX3基因编辑细胞作为供体细胞生产体细胞克隆猪，平均产活仔数显著减少，平均死胎数显著增加。此外，存活的IRX3体细胞克隆猪出生体重显著降低，活力较差，所有仔猪出生后不久死亡。因此，本研究的初步结果表明，IRX3在猪中可能具有重要生物学功能，不应将其作为降低巴马香猪脂肪含量的基因编辑靶点。此外，本研究表明敲除IRX3不利于猪的存活，在治疗人类肥胖中对IRX3进行靶向调控是否也会引发严重不良后果，还需要进一步研究。
