The genetically modified human foreskin fibroblast cell line (YhFF#8) stably expressing Cas9 gene: A lab resource report
Abstract 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 red...
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| Format: | Article |
| Language: | English |
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Shahid Sadoughi University of Medical Sciences
2024-02-01
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| Series: | International Journal of Reproductive BioMedicine |
| Subjects: | |
| Online Access: | https://doi.org/10.18502/ijrm.v22i1.15243 |
| _version_ | 1797258572645531648 |
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| author | Farzad Soheilipour Sohrab Boozarpour Shiva Aghaei Ehsan Farashahi Yazd |
| author_facet | Farzad Soheilipour Sohrab Boozarpour Shiva Aghaei Ehsan Farashahi Yazd |
| author_sort | Farzad Soheilipour |
| collection | DOAJ |
| description | Abstract 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. |
| first_indexed | 2024-04-24T22:55:40Z |
| format | Article |
| id | doaj.art-1a5b013c8f904011b6d5aeeec7b1248a |
| institution | Directory Open Access Journal |
| issn | 2476-4108 2476-3772 |
| language | English |
| last_indexed | 2024-04-24T22:55:40Z |
| publishDate | 2024-02-01 |
| publisher | Shahid Sadoughi University of Medical Sciences |
| record_format | Article |
| series | International Journal of Reproductive BioMedicine |
| spelling | doaj.art-1a5b013c8f904011b6d5aeeec7b1248a2024-03-18T05:27:14ZengShahid Sadoughi University of Medical SciencesInternational Journal of Reproductive BioMedicine2476-41082476-37722024-02-01221616810.18502/ijrm.v22i1.15243ijrm.v22i1.15243The genetically modified human foreskin fibroblast cell line (YhFF#8) stably expressing Cas9 gene: A lab resource reportFarzad Soheilipour0Sohrab Boozarpour1Shiva Aghaei2Ehsan Farashahi Yazd3 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. Stem Cell Biology Research Center, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran. Stem Cell Biology Research Center, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.Abstract 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.https://doi.org/10.18502/ijrm.v22i1.15243fibroblasts cell line genetic transduction crispr-cas9. |
| spellingShingle | Farzad Soheilipour Sohrab Boozarpour Shiva Aghaei Ehsan Farashahi Yazd The genetically modified human foreskin fibroblast cell line (YhFF#8) stably expressing Cas9 gene: A lab resource report International Journal of Reproductive BioMedicine fibroblasts cell line genetic transduction crispr-cas9. |
| title | The genetically modified human foreskin fibroblast cell line (YhFF#8) stably expressing Cas9 gene: A lab resource report |
| title_full | The genetically modified human foreskin fibroblast cell line (YhFF#8) stably expressing Cas9 gene: A lab resource report |
| title_fullStr | The genetically modified human foreskin fibroblast cell line (YhFF#8) stably expressing Cas9 gene: A lab resource report |
| title_full_unstemmed | The genetically modified human foreskin fibroblast cell line (YhFF#8) stably expressing Cas9 gene: A lab resource report |
| title_short | The genetically modified human foreskin fibroblast cell line (YhFF#8) stably expressing Cas9 gene: A lab resource report |
| title_sort | genetically modified human foreskin fibroblast cell line yhff 8 stably expressing cas9 gene a lab resource report |
| topic | fibroblasts cell line genetic transduction crispr-cas9. |
| url | https://doi.org/10.18502/ijrm.v22i1.15243 |
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