Production of CFTR Mutant Gene Model by Homologous Recombination System
Objective: The most common mutation in cystic fibrosis (CF), (ΔF508-CFTR), results in impaired protein maturation,folding and transportation to the surface of the cell. As a consequence of impaired protein maturation and/or transport from the extracellular matrix to the cell, different systems are i...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
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Royan Institute (ACECR), Tehran
2022-10-01
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| Series: | Cell Journal |
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| Online Access: | https://www.celljournal.org/article_253364_7998671eee9511a8abcb876a88e8d55e.pdf |
| _version_ | 1828344231023869952 |
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| author | Hanieh Rezaee mohammad Salehi Mojgan Bandehpour Sima Kalantari Sara Hosseini Khosrow Agin Bahram Kazemi |
| author_facet | Hanieh Rezaee mohammad Salehi Mojgan Bandehpour Sima Kalantari Sara Hosseini Khosrow Agin Bahram Kazemi |
| author_sort | Hanieh Rezaee |
| collection | DOAJ |
| description | Objective: The most common mutation in cystic fibrosis (CF), (ΔF508-CFTR), results in impaired protein maturation,folding and transportation to the surface of the cell. As a consequence of impaired protein maturation and/or transport from the extracellular matrix to the cell, different systems are influenced, including gastrointestinal system and glandular system, reproductive system and respiratory systems. CF models are essential tools to provide further knowledge ofCF pathophysiology. With this aim, we designed a transgenic CF model based on the homologous recombination (HR)system.Materials and Methods: In this experimental study, a specifically designed construct containing the CFTR gene with F508del was cloned into a PTZ57R cloning vector and then the construct was transformed into the male pronucleus by microinjection after in vitro fertilization (IVF). Then the rates of blastocyst formation and embryonic development at72 hours after IVF, were evaluated using the inverted microscope and the insertion of the construct was approved bypolymerase chain reaction (PCR) method.Results: The CFTR gene was successfully cloned into the PTZ57R cloning vector and overall, from 22 injected cells, 5 blastocysts were observed after pronuclear injection of the CFTR gene construct. PCR verification of the blastocyst with CFTR-specific primers represented complete recombination of CFTR into the mouse genome.Conclusion: For the first time we designed a unique genome construction that can be detected using a simple PCR method. The pronuclear injection was performed for the transformation of the genome construct into the male pronuclei using microinjection and the development of zygote to the blastocyst stage has been observed following transgenesis. |
| first_indexed | 2024-04-13T23:54:24Z |
| format | Article |
| id | doaj.art-4c00156b9bcf48c5b2fa38de1b7fc93e |
| institution | Directory Open Access Journal |
| issn | 2228-5806 2228-5814 |
| language | English |
| last_indexed | 2024-04-13T23:54:24Z |
| publishDate | 2022-10-01 |
| publisher | Royan Institute (ACECR), Tehran |
| record_format | Article |
| series | Cell Journal |
| spelling | doaj.art-4c00156b9bcf48c5b2fa38de1b7fc93e2022-12-22T02:23:56ZengRoyan Institute (ACECR), TehranCell Journal2228-58062228-58142022-10-01241059660210.22074/cellj.2022.8408253364Production of CFTR Mutant Gene Model by Homologous Recombination SystemHanieh Rezaee0mohammad Salehi1Mojgan Bandehpour2Sima Kalantari3Sara Hosseini4Khosrow Agin5Bahram Kazemi6Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, IranDepartment of Medical Biotechnology, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, IranCellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, IranDepartment of Medical Biotechnology, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, IranMom Fertility and Infertility Research and Innovation Center, Tehran, IranToxicological Research Center, Loghman-Hakim Hospital, Department of Clinical Toxicology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, IranDepartment of Medical Biotechnology, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, IranObjective: The most common mutation in cystic fibrosis (CF), (ΔF508-CFTR), results in impaired protein maturation,folding and transportation to the surface of the cell. As a consequence of impaired protein maturation and/or transport from the extracellular matrix to the cell, different systems are influenced, including gastrointestinal system and glandular system, reproductive system and respiratory systems. CF models are essential tools to provide further knowledge ofCF pathophysiology. With this aim, we designed a transgenic CF model based on the homologous recombination (HR)system.Materials and Methods: In this experimental study, a specifically designed construct containing the CFTR gene with F508del was cloned into a PTZ57R cloning vector and then the construct was transformed into the male pronucleus by microinjection after in vitro fertilization (IVF). Then the rates of blastocyst formation and embryonic development at72 hours after IVF, were evaluated using the inverted microscope and the insertion of the construct was approved bypolymerase chain reaction (PCR) method.Results: The CFTR gene was successfully cloned into the PTZ57R cloning vector and overall, from 22 injected cells, 5 blastocysts were observed after pronuclear injection of the CFTR gene construct. PCR verification of the blastocyst with CFTR-specific primers represented complete recombination of CFTR into the mouse genome.Conclusion: For the first time we designed a unique genome construction that can be detected using a simple PCR method. The pronuclear injection was performed for the transformation of the genome construct into the male pronuclei using microinjection and the development of zygote to the blastocyst stage has been observed following transgenesis.https://www.celljournal.org/article_253364_7998671eee9511a8abcb876a88e8d55e.pdfanimal modelcystic fibrosishomologous recombinationpolymerase chain reaction |
| spellingShingle | Hanieh Rezaee mohammad Salehi Mojgan Bandehpour Sima Kalantari Sara Hosseini Khosrow Agin Bahram Kazemi Production of CFTR Mutant Gene Model by Homologous Recombination System Cell Journal animal model cystic fibrosis homologous recombination polymerase chain reaction |
| title | Production of CFTR Mutant Gene Model by Homologous Recombination System |
| title_full | Production of CFTR Mutant Gene Model by Homologous Recombination System |
| title_fullStr | Production of CFTR Mutant Gene Model by Homologous Recombination System |
| title_full_unstemmed | Production of CFTR Mutant Gene Model by Homologous Recombination System |
| title_short | Production of CFTR Mutant Gene Model by Homologous Recombination System |
| title_sort | production of cftr mutant gene model by homologous recombination system |
| topic | animal model cystic fibrosis homologous recombination polymerase chain reaction |
| url | https://www.celljournal.org/article_253364_7998671eee9511a8abcb876a88e8d55e.pdf |
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