Volume 29, Issue 1 (3-2026)
J Arak Uni Med Sci 2026, 29(1): 19-25 |
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Faghihi F, Amani J, Gholami M, salahshourifar I. Construction and Cloning of CRISPR /Cas9 Target Gene and Confirmation of Cell Line Identity by QF - PCR. J Arak Uni Med Sci 2026; 29 (1) :19-25
URL: http://jams.arakmu.ac.ir/article-1-8269-en.html
URL: http://jams.arakmu.ac.ir/article-1-8269-en.html
1- PhD Student, Department of Biology, SR.C., Islamic Azad University, Tehran, Iran
2- Professor, Applied Microbiology Research Center, Biomedicine Technologies Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran ,jafar.amani@gmail.com
3- Associate Professor, Department of Biochemistry and Genetics, School of Medicine, Arak University of Medical Sciences, Arak, Iran
4- Assistant Professor, Department of Biology, SR.C., Islamic Azad University, Tehran, Iran
2- Professor, Applied Microbiology Research Center, Biomedicine Technologies Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran ,
3- Associate Professor, Department of Biochemistry and Genetics, School of Medicine, Arak University of Medical Sciences, Arak, Iran
4- Assistant Professor, Department of Biology, SR.C., Islamic Azad University, Tehran, Iran
Abstract: (645 Views)
Introduction: Autism Spectrum Disorder (ASD) and CDKL5 deficiency disorder are among the nervous and developmental disorder with genetic origin that requires accurate molecular models are required to study the mechanisms of disease and develop new treatments. The CRISPR / Cas9 genome editing system is an efficient tool to generate targeted changes in the genome. The aim of this study was to design and construct CRISPR / Cas9 vector carrier sgRNA for CDKL5 gene and to confirm the cloning and identity of the cell line used by molecular methods.
Methods: In this experimental study, at first, specific primers were designed for region in the cdkl5 gene. Oligo nouclotide forward and reverse annealing and cloned in PX458 vector. After transformation of E. coli strains DH5, positive colonies were confirmed by PCR and sequencing methods. Also to ensure the identity of the HEK293T cell line, QF - PCR was used to determine the sex and chromosome study.
Results: Colony-PCR results showed the expected band of 230 bp in selected colonies. Sequencing also confirmed insert sgRNA in the vector. The results of QF-PCR demonstrated specific peaks patterns for sex chromosomes that confirmed cell line identity.
Conclusions: The CRISPR / Cas9 target gene expression was successfully performed and molecular approvals were obtained for cloning and analysis of the cell line. This vector is proposed for further studies on gene expression in cellular models.
Methods: In this experimental study, at first, specific primers were designed for region in the cdkl5 gene. Oligo nouclotide forward and reverse annealing and cloned in PX458 vector. After transformation of E. coli strains DH5, positive colonies were confirmed by PCR and sequencing methods. Also to ensure the identity of the HEK293T cell line, QF - PCR was used to determine the sex and chromosome study.
Results: Colony-PCR results showed the expected band of 230 bp in selected colonies. Sequencing also confirmed insert sgRNA in the vector. The results of QF-PCR demonstrated specific peaks patterns for sex chromosomes that confirmed cell line identity.
Conclusions: The CRISPR / Cas9 target gene expression was successfully performed and molecular approvals were obtained for cloning and analysis of the cell line. This vector is proposed for further studies on gene expression in cellular models.
Type of Study: Original Atricle |
Subject:
Basic Sciences
Received: 2026/03/13 | Accepted: 2026/03/16
Received: 2026/03/13 | Accepted: 2026/03/16
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