The Relationship between Embryonic Development and the Efficiency of Target Mutations in Porcine Endogenous Retroviruses (PERVs) Pol Genes in Porcine Embryos
The Relationship between Embryonic Development and the Efficiency of Target Mutations in Porcine Endogenous Retroviruses (PERVs) Pol Genes in Porcine Embryos
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DOI：
10.3390/ani9090593
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发表时间：
2019-09-01
期刊：
影响因子：
3
通讯作者：
Otoi, Takeshige
中科院分区：
文献类型：
--
作者：
Hirata, Maki;Wittayarat, Manita;Otoi, Takeshige
简单总结 猪内源性逆转录病毒（PERV）失活的猪是异种移植的首选供体来源，因为 PERV 可能成为接受猪器官异种移植的人类的感染性病原体。然而，据报道，猪细胞中 PERV pol 基因的失活会影响细胞生长。本研究阐明了猪胚胎中PERV pol基因的突变与其发育之间的关系。使用 Cas9 蛋白 (GEEP) 系统的电穿孔进行基因组编辑，将三种不同的针对 PERV pol 基因的 gRNA 引入猪受精卵中。结果表明，猪胚胎PERV pol基因的目标突变效率与胚胎发育之间存在负相关关系。猪内源性逆转录病毒（PERV）是在猪基因组中发现的一种原病毒，可能在接受猪器官异种移植的人类中充当感染性病原体。据报道，猪细胞中 PERV pol 基因的失活会影响细胞生长。因此，利用基因组编辑技术对猪胚胎中PERV pol基因进行突变可能会影响胚胎发育。本研究旨在探讨猪胚胎中PERV pol基因的突变与其发育之间的关系。我们使用 Cas9 蛋白 (GEEP) 系统的电穿孔进行基因组编辑，将三种不同的 gRNA（gRNA1、2 和 3）单独或组合引入猪受精卵中。所有三种 gRNA 均以 PERV pol 基因为目标，我们评估了它们对猪胚胎发育的影响。我们的结果表明，单独和联合使用 gRNA3 电穿孔的受精卵的囊胚形成率显着低于使用 gRNA1 电穿孔的受精卵（p < 0.05）。在 2 至 8 细胞阶段的单个囊胚和混合胚胎中，通过 PERV pol 基因靶位点测序评估的突变率在三种 gRNA 之间没有差异。然而，在单独使用 gRNA3 和组合使用 gRNA3 进行电穿孔的胚胎中，2 至 8 细胞阶段的突变胚胎中插入缺失突变的频率趋于较高。胚胎发育可能会受到诱导高频插入缺失突变的 gRNA 的影响。
Simple Summary Pigs with porcine endogenous retrovirus (PERV) inactivation are preferable donor sources for xenotransplantation because the PERV may act as an infectious pathogen for humans who receive pig organ xenotransplantation. However, inactivation of the PERV pol gene in porcine cells reportedly affects cell growth. The present study clarified the relationship between the mutation of the PERV pol gene in porcine embryos and their development. Three different gRNAs targeting the PERV pol gene were introduced into porcine zygotes by genome editing using electroporation of the Cas9 protein (GEEP) system. The results demonstrated a negative relationship between the embryonic development and the efficiency of target mutations in the PERV pol gene of the porcine embryos. Porcine endogenous retrovirus (PERV) is a provirus found in the pig genome that may act as an infectious pathogen in humans who receive pig organ xenotransplantation. Inactivation of the PERV pol gene in porcine cells reportedly affects cell growth. Therefore, the mutation of PERV pol gene in porcine embryos using genome editing may affect the embryonic development. The present study was carried out to investigate the relationship between the mutation of the PERV pol gene in porcine embryos and their development. We introduced, either alone or in combination, three different gRNAs (gRNA1, 2, and 3) into porcine zygotes by genome editing using electroporation of the Cas9 protein (GEEP) system. All three gRNAs targeted the PERV pol gene, and we assessed their effects on porcine embryonic development. Our results showed that the blastocyst formation rates of zygotes electroporated with gRNA3-alone and in combination-were significantly lower (p < 0.05) than those of zygotes electroporated with gRNA1. The mutation rates assessed by the PERV pol gene target site sequencing in individual blastocysts and pooled embryos at the 2-to-8-cell stage did not differ among the three gRNAs. However, the frequency of indel mutations in mutant embryos at the 2-to-8-cell stage trended higher in the embryos electroporated with gRNA3 alone and in combination. Embryonic development may be affected by gRNAs that induce high-frequency indel mutations.