Dual selection vector (pDC459) comprises more than one selectable and counter-selectable markers for improved clustered regularly interspaced short palindromic repeats/Cas9 gene editing, where the selectable markers are an ampicillin and a puromycin resistance gene
	VERMA P; KUMAR V; CHAUHAN S S
	2023-09-20
专利权人	ALL INDIA INST MEDICAL SCI (ALLI-Non-standard)
申请日期	2023-09-20
专利号	IN202311063385-A
成果简介	NOVELTY - Dual selection vector (pDC459) comprises more than one selectable and counter-selectable markers for improved clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) gene editing, where the selectable markers are an ampicillin and a puromycin resistance gene which are used for selecting bacterial cells and mammalian cells respectively and the counter selectable marker is PIGS knockout gene. USE - Dual selection vector (pDC459). ADVANTAGE - The dual selection vector (pDC459) help in editing in human cell lines, and knockout of two genes simultaneously or in deletions of gene fragments by using two gRNAs. DETAILED DESCRIPTION - An INDEPENDENT CLAIM is included for method for obtaining the vector pDC459, which involves: (1) developing of eukaryotic cell culture selected from SCC4, HERK293T, DOK, AW5805, LN428 and U87, preferably SCC4 and HERK293T cell lines, followed by sub-culturing of eukaryotic cells by trypsinization and freezing of cells; (2) targeting the eukaryotic cell culture and cloning two gRNA's exon1 and exon2 of PIGS gene under gRNA scaffold in a pDG459 plasmid for knockout of PIGS gene as a counter selectable maker; (3) performing polymerse chain reaction (PCR) amplification, followed by restriction digesting the knockout plasmid obtained; (4) DNA ligating the plasmid obtained to cloning plasmid DNA; (5) screening of recombinant clones obtained by restriction analysis, PCR, designing overlaps primer, in-fusion reaction; and (6) cloning two empty gRNAs cassettes under individual U6 promoters in the recombinant clone obtained and the resulting construct was named as pDC459.
IPC 分类号	C12N-015/113 ; C12N-015/65 ; C12N-015/85 ; C12N-015/90 ; C12N-009/22
国家	印度
专业领域	医药卫生
语种	英语
成果类型	专利
文献类型	科技成果
条目标识符	http://119.78.100.226:8889/handle/3KE4DYBR/20310
专题	中国科学院新疆生态与地理研究所
作者单位	ALL INDIA INST MEDICAL SCI (ALLI-Non-standard)
推荐引用方式 GB/T 7714	VERMA P,KUMAR V,CHAUHAN S S. Dual selection vector (pDC459) comprises more than one selectable and counter-selectable markers for improved clustered regularly interspaced short palindromic repeats/Cas9 gene editing, where the selectable markers are an ampicillin and a puromycin resistance gene. IN202311063385-A[P]. 2023.

条目包含的文件
条目无相关文件。

除非特别说明，本系统中所有内容都受版权保护，并保留所有权利。