International Journal of
Reproductive Biomedicine
Thu, Nov 21, 2024
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Volume 22, Issue 1 (January 2024)
IJRM 2024, 22(1): 61-68 |
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Ethics code: IR.SSU.REC.1400.139
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Soheilipour F, Boozarpour S, Aghaei S, Farashahi Yazd E. The genetically modified human foreskin fibroblast cell line (YhFF#8) stably expressing Cas9 gene: A lab resource report. IJRM 2024; 22 (1) :61-68
URL: http://ijrm.ir/article-1-3175-en.html
URL: http://ijrm.ir/article-1-3175-en.html
1- Stem Cell Biology Research Center, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran. Department of Biology, Faculty of Basic Sciences, Gonbad Kavous University, Gonbad Kavous, Iran.
2- Department of Biology, Faculty of Basic Sciences, Gonbad Kavous University, Gonbad Kavous, Iran.
3- Stem Cell Biology Research Center, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
4- Stem Cell Biology Research Center, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran. ,ehsanfarashahi@ssu.ac.ir
2- Department of Biology, Faculty of Basic Sciences, Gonbad Kavous University, Gonbad Kavous, Iran.
3- Stem Cell Biology Research Center, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
4- Stem Cell Biology Research Center, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran. ,
Abstract: (323 Views)
Background: Stable Cas9 (CRISPR-associated protein 9)-expressing cell lines have emerged as valuable tools in genetic research, enhancing the efficiency of the CRISPR/Cas9 system and streamlining gene editing procedures. These cell lines enable simultaneous editing of multiple genes and reduce the overall editing time.
Objective: This study aimed to develop a stable human fibroblast cell line capable of genetic conversion into a mutant form, serving as a cellular model for a specific genetic disease. The established cell line facilitates investigation of disease mechanisms, testing of potential treatments, and gaining insights into underlying molecular processes.
Materials and Methods: Human embryonic kidney 293LTV cells were used to produce pseudo-virus particles, while Yazd human foreskin fibroblasts batch 8 (YhFF#8) cells were targeted for genetic modification. Transfection of human embryonic kidney 293LTV cells with pCDH-Cas9 plasmid DNA generated pseudo-viral particles. YhFF#8 cells were transduced and selected using antibiotics. Green fluorescent protein (GFP) detection confirmed successful transduction and selection. Relative expression levels of the Cas9 gene were determined by quantitative polymerase chain reaction.
Results: The study validated the fidelity of the Cas9 gene cassette sequence and its transcriptional activity. Transduced YhFF#8 cells exhibited green fluorescence, with antibiotic selection resulting in nearly 100% transduced cells. A reporter GFP gene enabled real-time monitoring of YhFF#8-Cas9-GFP-PuroR cells using fluorescence microscopy.
Conclusion: YhFF#8-Cas9-GFP-PuroR cells, labeled and susceptible to genomic editing, provide an optimal source for generating induced pluripotent stem cell lines for future biomedical research.
Objective: This study aimed to develop a stable human fibroblast cell line capable of genetic conversion into a mutant form, serving as a cellular model for a specific genetic disease. The established cell line facilitates investigation of disease mechanisms, testing of potential treatments, and gaining insights into underlying molecular processes.
Materials and Methods: Human embryonic kidney 293LTV cells were used to produce pseudo-virus particles, while Yazd human foreskin fibroblasts batch 8 (YhFF#8) cells were targeted for genetic modification. Transfection of human embryonic kidney 293LTV cells with pCDH-Cas9 plasmid DNA generated pseudo-viral particles. YhFF#8 cells were transduced and selected using antibiotics. Green fluorescent protein (GFP) detection confirmed successful transduction and selection. Relative expression levels of the Cas9 gene were determined by quantitative polymerase chain reaction.
Results: The study validated the fidelity of the Cas9 gene cassette sequence and its transcriptional activity. Transduced YhFF#8 cells exhibited green fluorescence, with antibiotic selection resulting in nearly 100% transduced cells. A reporter GFP gene enabled real-time monitoring of YhFF#8-Cas9-GFP-PuroR cells using fluorescence microscopy.
Conclusion: YhFF#8-Cas9-GFP-PuroR cells, labeled and susceptible to genomic editing, provide an optimal source for generating induced pluripotent stem cell lines for future biomedical research.
Type of Study: Original Article |
Subject:
Stem Cell & Cloning
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