教員業績データベース |
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論文種別 | 原著 |
言語種別 | 英語 |
査読の有無 | 査読あり |
表題 | Knockout of Cytidine Monophospho-N-Acetylneuraminic Acid (CMP-NeuAc) Hydroxylase From Porcine Endothelial Cells by a CRISPR System |
掲載誌名 | 正式名:Transplantation proceedings 略 称:Transplant Proc ISSNコード:1873-2623(Electronic)0041-1345(Linking) |
掲載区分 | 国外 |
巻・号・頁 | 48(4),pp.1320-2 |
著者・共著者 | 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 |
発行年月 | 2016/05 |
概要 | BACKGROUND: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).METHODS: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.RESULTS: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.CONCLUSIONS:Changes in the expressionCONCLUSIONS: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. |
DOI | 10.1016/j.transproceed.2015.10.065 |
PMID | 27320613 |