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Generation of K562 cell line expressing Cas9 endonuclease (CRISPR-associated9)
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F. Ensari   , M. Nikougoftar  , A.A. Hamidieh , M. Shamsara |
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| Keywords: Key words: Cell Line, Electroporation, Gene Editing |
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| Abstract (HTML) (4010 Views)
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Type of Study: Research |
Subject:
Biotechnology
Published: 2019/04/8
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Full-Text: (2421 Views) |
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Sci J Iran Blood Transfus Organ 2018; 16(4): 32-43
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Generation of K562 cell line expressing Cas9
endonuclease (CRISPR-associated9)
Ansari F.1, Nikougoftar Zarif M.1, Hamidieh A.A.2, Shamsara M.3
1Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran
2Pediatric Stem Cell Transplantation Department, Children’s Medical Center, Tehran University of Medical Science, Tehran, Iran
3National Institute of Genetic Engineering and Biotechnology, Tehran, Iran
Abstract
Background and Objectives
The genome of cell lines is nowadays edited to create disease models and treat them. Of course, the size of the cas9 gene has caused problems like the low efficiency of the CRISPR system. To solve this problem, Cas9 expressing cell lines have been generated in which CRISPR RNA should only be transfected to the cell.
Materials and Methods
This article is experimental. PGK-PURO / CMV (PPC) fragment was amplified with PCR from pAAVS1-puro-DNR vector and cloned in pTG19-T vector. The PPC fragment from this vector was removed by KpnI and EcoRI enzymes. Also the pCas-Guide-AAVS1 vector was subjected to the same enzymatic cutting and its attachment to the PPC fragment resulted in the production of the pPPC-Cas vector. After optimizing the electroporation conditions, the pPPC-Cas vector was electroporated into K562 cells and puromycin -resistant cells were selected and Cas9 expression level was evaluated by Real-time PCR.
Results
A PCR fragment of 2514 bp was amplified. The vector pPPC-Cas was cloned in two steps. puromycin -resistant transfected cells were selected. Clonal selection was carried out and three colonies with high, medium and low expression level of Cas9 were isolated.
Conclusions
The Cas9-expressing K562 cells derived in this study can be applied both for functional genomic researches and design cellular models of human diseases in future.
Key words: Cell Line, Electroporation, Gene Editing
Received: 3 Sep 2018
Accepted: 15 Jan 2019
Correspondence: Shamsara M., PhD in Genetics. Assistant Professor of Agricultural Biotechnology, National Institute of Genetic Engineering and Biotechnology.
P.O.Box: 14965-161, Tehran, Iran. Tel: (+9821) 44787414; Fax: (+9821) 44787399
E-mail: shamsa@nigeb.ac.ir
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Ensari F, Nikougoftar M, Hamidieh A, Shamsara M. Generation of K562 cell line expressing Cas9 endonuclease (CRISPR-associated9). Sci J Iran Blood Transfus Organ 2019; 16 (1) :32-43 URL: http://bloodjournal.ir/article-1-1223-en.html
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