Volume 16, Issue 1 (Spring 2019)
Sci J Iran Blood Transfus Organ 2019, 16(1): 32-43 |
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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
URL: http://bloodjournal.ir/article-1-1223-en.html
Abstract: (4429 Views)
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.
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.
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