Sakai R, Esaki Y, Hasuwa H, Ikawa M, Lo P, Matsuura R, Nakahata K, Zenitani M, Asada M, Maeda A, Eguchi H, Okuyama H, Miyagawa S
Department of Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan; and Research Institute for Microbial Diseases, Osaka University, Osaka, Japan.
Department of Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan; and Research Institute for Microbial Diseases, Osaka University, Osaka, Japan.
Transplant Proc. 2016 May;48(4):1320-2. doi: 10.1016/j.transproceed.2015.10.065.
We attempted to knock out the expression of Hanganutziu-Deicher (H-D) antigens through the use of a CRISPR (clustered regulatory interspaced short palindromic repeat)/Cas9 system for pig cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH).
Plasmids expressing hCas9 and sgRNA for pCMAH were prepared by ligating oligos into the BbsI site of pX330. The N-terminal and C-terminal EGFP coding regions overlapping 482 bp were PCR-amplified and placed under a ubiquitous CAG promoter. The approximately 400-bp genomic fragments containing the sgRNA target sequence of pCMAH were placed into the multi-cloning sites flanked by the EGFP fragments. The pCAG-EGxxFP-target was mixed with pX330 with/without the sgRNA sequences and then introduced into HEK293T cells.
Four oligos and primers, gSO1, gSO3, gSO4, and gSO8, were nominated from 8 candidates. Among them, gSO1 showed the best efficiency. Pig endothelial cells (PECs) from an α-Gal knockout pig were then used to examine the changes in the expression of the H-D antigen by the knockout of the CMAH genome by the pX330-gS01.
Changes in the expression of the H-D antigen in the PECs with the CRISPR (gS01) were clear in comparison with those in the parental cells, on the basis of FACS analysis data. The expression of the H-D antigen can be knocked out by use of the CRISPR system for pCMAH, thus confirming that this system is a very convenient system for producing knockout pigs.
我们试图通过使用针对猪胞苷单磷酸-N-乙酰神经氨酸羟化酶（CMAH）的CRISPR（成簇规律间隔短回文重复序列）/Cas9系统来敲除汉努齐乌-戴歇尔（H-D）抗原的表达。
通过将寡核苷酸连接到pX330的BbsI位点，制备用于pCMAH的表达hCas9和sgRNA的质粒。对重叠482 bp的N端和C端EGFP编码区进行PCR扩增，并置于普遍存在的CAG启动子之下。将包含pCMAH的sgRNA靶序列的约400 bp基因组片段置于由EGFP片段侧翼的多克隆位点中。将pCAG-EGxxFP-靶标与带有/不带有sgRNA序列的pX330混合，然后导入HEK293T细胞。
从8个候选物中选出了4个寡核苷酸和引物，即gSO1、gSO3、gSO4和gSO8。其中，gSO1显示出最佳效率。然后使用来自α-Gal基因敲除猪的猪内皮细胞（PEC），通过pX330-gS01敲除CMAH基因组来检测H-D抗原表达的变化。
基于流式细胞术分析数据，与亲代细胞相比，用CRISPR（gS01）处理的PEC中H-D抗原的表达变化明显。使用针对pCMAH的CRISPR系统可以敲除H-D抗原的表达，从而证实该系统是生产基因敲除猪的非常方便的系统。
