Disabling of nephrogenesis in porcine embryos via CRISPR/Cas9-mediated SIX1 and SIX4 gene targeting
Disabling of nephrogenesis in porcine embryos via CRISPR/Cas9-mediated SIX1 and SIX4 gene targeting
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DOI：
10.1111/xen.12484
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发表时间：
2019-05-01
影响因子：
3.9
通讯作者：
Li, Rongfeng
中科院分区：
文献类型：
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作者：
Wang, Junzheng;Liu, Manling;Li, Rongfeng
SIX1和SIX4基因在肾脏发育中起重要作用。我们通过CRISPR/Cas9和体细胞核移植产生SIX1(-/-)和SIX1(-/-)/SIX4(-/-)猪胚胎，评价了这些基因对猪肾脏发育的影响。获得3个SIX1(-/-)胚胎和16个不同发育阶段的SIX1(-/-)/SIX4(-/-)胎儿。SIX1(-/-)胎儿表现为左肾移行阻塞，双肾体积较小。输尿管芽不能形成正常的分支和收集系统。肾脏发育相关基因的异常表达(PAX2、PAX8和BMP4下调，EYA1和SALL1上调)也在SIX1(-/-)胎肾中观察到，并在体外证实了SIX1基因缺失后在猪肾上皮细胞(PK15)中的表达。SIX1(-/-)/SIX4(-/-)的胎儿表现出更严重的表型，大多数胎儿在妊娠早期表现出发育迟缓。肾脏仅发育到后肾形成的初始阶段。这些结果表明，SIX1和SIX4是猪后肾发育的关键基因。创造肾功能不全的猪胚胎为利用囊胚互补在猪体内生成人肾提供了一个平台。
SIX1 and SIX4 genes play critical roles in kidney development. We evaluated the effect of these genes on pig kidney development by generating SIX1(-/-) and SIX1(-/-)/SIX4(-/-) pig foetuses using CRISPR/Cas9 and somatic cell nuclear transfer. We obtained 3 SIX1(-/-) foetuses and 16 SIX1(-/-)/SIX4(-/-) foetuses at different developmental stages. The SIX1(-/-) foetuses showed a migration block of the left kidney and a smaller size for both kidneys. The ureteric bud failed to form the normal branching and collecting system. Abnormal expressions of kidney development-related genes (downregulation of PAX2, PAX8, and BMP4 and upregulation of EYA1 and SALL1) were also observed in SIX1(-/-) foetal kidneys and confirmed in vitro in porcine kidney epithelial cells (PK15) following SIX1 gene deletion. The SIX1(-/-)/SIX4(-/-) foetuses exhibited more severe phenotypes, with most foetuses showing retarded development at early stages of gestation. The kidney developed only to the initial stage of metanephros formation. These results demonstrated that SIX1 and SIX4 are key genes for porcine metanephros development. The creation of kidney-deficient porcine foetuses provides a platform for generating human kidneys inside pigs using blastocyst complementation.