Efficient Generation of an Fah/Rag2 Dual-Gene Knockout Porcine Cell Line Using CRISPR/Cas9 and Adenovirus
Efficient Generation of an Fah/Rag2 Dual-Gene Knockout Porcine Cell Line Using CRISPR/Cas9 and Adenovirus
复制标题
使用 CRISPR/Cas9 和腺病毒高效生成 Fah/Rag2 双基因敲除猪细胞系
DOI：
10.1089/dna.2018.4493
复制
发表时间：
2019-04-01
影响因子：
3.1
通讯作者：
Bu, Hong
中科院分区：
文献类型：
--
作者：
Gao, Mengyu;Zhang, Bingqi;Bu, Hong
人肝细胞的短缺仍然是肝细胞移植和生物人工肝（BAL）支持治疗广泛应用的一个重要限制。重组激活基因2（Rag 2）和富马酰乙酰乙酸水解酶（Fah）缺陷小鼠可以高度重新填充与人肝细胞。然而，Fah/Rag 2缺陷小鼠每只小鼠只能产生至多1 x 10（8）个人肝细胞。我们假设每头Fah/Rag 2缺陷猪可以产生2-10 x 10（10）个人肝细胞，这是肝细胞移植和BAL治疗的充足供应。在一种新的方法中，我们使用稳定转染的Cas9细胞和含有荧光报告基因的单向导RNA腺病毒来富集具有Fah/Rag 2双基因突变的猪细胞。这导致构建Fah/Rag 2双敲除猪髂动脉内皮细胞，其随后用于产生Fah/Rag 2缺陷型猪。
The shortage of human hepatocytes continues to be a significant limitation for the widespread application of hepatocyte transplantation and bioartificial liver (BAL) support therapy. Recombinant activation gene 2 (Rag2) and fumarylacetoacetate hydrolase (Fah)-deficient mice could be highly repopulated with human hepatocytes. However, Fah/Rag2-deficient mice can only produce up to 1 x 10(8) human hepatocytes per mouse. We hypothesized that 2-10 x 10(10) human hepatocytes can be produced per Fah/Rag2-deficient pig, which is an adequate supply for hepatocyte transplantation and BAL therapy. In a novel approach, we used stably transfected Cas9 cells and single-guide RNA adenoviruses containing fluorescent reporters to enrich porcine cells with Fah/Rag2 dual gene mutations. This resulted in the construction of Fah/Rag2 double knockout porcine iliac artery endothelial cells, which were subsequently used for generating Fah/Rag2-deficient pigs.