2016年5月
Knockout of Cytidine Monophospho-N-Acetylneuraminic Acid (CMP-NeuAc) Hydroxylase From Porcine Endothelial Cells by a CRISPR System
TRANSPLANTATION PROCEEDINGS
- 巻
- 48
- 号
- 4
- 開始ページ
- 1320
- 終了ページ
- 1322
- 記述言語
- 英語
- 掲載種別
- 記事・総説・解説・論説等(国際会議プロシーディングズ)
- DOI
- 10.1016/j.transproceed.2015.10.065
- 出版者・発行元
- ELSEVIER SCIENCE INC
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 alpha-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-gSO1.
Conclusions. Changes in the expression of the H-D antigen in the PECs with the CRISPR (gSO1) 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.
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 alpha-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-gSO1.
Conclusions. Changes in the expression of the H-D antigen in the PECs with the CRISPR (gSO1) 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.
- リンク情報
- ID情報
-
- DOI : 10.1016/j.transproceed.2015.10.065
- ISSN : 0041-1345
- eISSN : 1873-2623
- Web of Science ID : WOS:000378760400074