The effect of CRISPR constructs microinjection on the expression of developmental genes in Rag1 knocked‐out mice embryo
Abstract Despite all the advances in the production of transgenic mice, the production efficiency of these animal models is still low. Given that the expression of developmental genes has a critical role in growth and development of embryo, we determined the expression pattern of pluripotency, troph...
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| Format: | Article |
| Language: | English |
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Wiley
2021-07-01
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| Series: | Veterinary Medicine and Science |
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| Online Access: | https://doi.org/10.1002/vms3.380 |
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| author | Maryam Salimi Abolfazl Shirazi Koushan Sineh Sepehr Mohsen Norouzian Vahid Ebrahimi Maryam Mehravar Mohammad Majidi Mohammad M. Mehrazar |
| author_facet | Maryam Salimi Abolfazl Shirazi Koushan Sineh Sepehr Mohsen Norouzian Vahid Ebrahimi Maryam Mehravar Mohammad Majidi Mohammad M. Mehrazar |
| author_sort | Maryam Salimi |
| collection | DOAJ |
| description | Abstract Despite all the advances in the production of transgenic mice, the production efficiency of these animal models is still low. Given that the expression of developmental genes has a critical role in growth and development of embryo, we determined the expression pattern of pluripotency, trophectoderm and imprinting genes in the Rag1 (recombination‐activating gene 1) knocked‐out blastocysts resulting from microinjection of CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR‐associated protein 9) constructs into the zygote cytoplasm of C57bl6 mice. Following microinjection, the embryos were cultured and the gene expression of developed blastocysts and natural blastocysts (Sham and control groups) were evaluated using real‐time PCR. The agarose gel to confirm the deletion in the Rag1 gene in Rag1 knocked‐out blastocyst. Our results showed that the expression of trophectoderm genes (‐TEAD‐4 and Cdx2), pluripotency genes (Nanog and Oct‐4) and imprinting gene (H19) in the Rag1 knocked‐out group was significantly lower compared with the embryos obtained from Natural fertilization. According to these findings, manipulation, embryo culture and microinjection of CRISPR constructs into the zygote cytoplasm of mice led to reduced expression of imprinting, pluripotency and trophectoderm genes. Therefore, the Rag1 knocked‐out embryos produced by the CRISPR/Cas9 system are of low quality, which reduces the chances of live birth in these animals and may cause various abnormalities in fetuses. |
| first_indexed | 2024-12-19T17:29:52Z |
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| id | doaj.art-12ea8adeeda0478a97e6d24f997d5998 |
| institution | Directory Open Access Journal |
| issn | 2053-1095 |
| language | English |
| last_indexed | 2024-12-19T17:29:52Z |
| publishDate | 2021-07-01 |
| publisher | Wiley |
| record_format | Article |
| series | Veterinary Medicine and Science |
| spelling | doaj.art-12ea8adeeda0478a97e6d24f997d59982022-12-21T20:12:28ZengWileyVeterinary Medicine and Science2053-10952021-07-01741436144210.1002/vms3.380The effect of CRISPR constructs microinjection on the expression of developmental genes in Rag1 knocked‐out mice embryoMaryam Salimi0Abolfazl Shirazi1Koushan Sineh Sepehr2Mohsen Norouzian3Vahid Ebrahimi4Maryam Mehravar5Mohammad Majidi6Mohammad M. Mehrazar7Department of Biology and Anatomical Sciences Faculty of Medicine Shahid Beheshti University of Medical Sciences Tehran IranReproductive Biotechnology Research Center Avicenna Research InstituteACECR Tehran IranLaboratory Sciences Research Center Golestan University of Medical Sciences Gorgan IranDepartment of Biology and Anatomical Sciences Faculty of Medicine Shahid Beheshti University of Medical Sciences Tehran IranDepartment of Anatomy and Cell Biology School of Medicine Mashhad University of Medical Sciences Mashhad IranDepartment of Gametes and Cloning Research Institute of Animal Embryo TechnologyShahrekord University Shahrekord IranDepartment of Tissue Engineering & Regenerative Medicine Faculty of Advanced Technologies in Medicine Iran University of Medical Sciences Tehran IranDepartment of Gametes and Cloning Research Institute of Animal Embryo TechnologyShahrekord University Shahrekord IranAbstract Despite all the advances in the production of transgenic mice, the production efficiency of these animal models is still low. Given that the expression of developmental genes has a critical role in growth and development of embryo, we determined the expression pattern of pluripotency, trophectoderm and imprinting genes in the Rag1 (recombination‐activating gene 1) knocked‐out blastocysts resulting from microinjection of CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR‐associated protein 9) constructs into the zygote cytoplasm of C57bl6 mice. Following microinjection, the embryos were cultured and the gene expression of developed blastocysts and natural blastocysts (Sham and control groups) were evaluated using real‐time PCR. The agarose gel to confirm the deletion in the Rag1 gene in Rag1 knocked‐out blastocyst. Our results showed that the expression of trophectoderm genes (‐TEAD‐4 and Cdx2), pluripotency genes (Nanog and Oct‐4) and imprinting gene (H19) in the Rag1 knocked‐out group was significantly lower compared with the embryos obtained from Natural fertilization. According to these findings, manipulation, embryo culture and microinjection of CRISPR constructs into the zygote cytoplasm of mice led to reduced expression of imprinting, pluripotency and trophectoderm genes. Therefore, the Rag1 knocked‐out embryos produced by the CRISPR/Cas9 system are of low quality, which reduces the chances of live birth in these animals and may cause various abnormalities in fetuses.https://doi.org/10.1002/vms3.380CRISPR constructsdevelopmental genesmicroinjectionRag1 knocked‐out embryos |
| spellingShingle | Maryam Salimi Abolfazl Shirazi Koushan Sineh Sepehr Mohsen Norouzian Vahid Ebrahimi Maryam Mehravar Mohammad Majidi Mohammad M. Mehrazar The effect of CRISPR constructs microinjection on the expression of developmental genes in Rag1 knocked‐out mice embryo Veterinary Medicine and Science CRISPR constructs developmental genes microinjection Rag1 knocked‐out embryos |
| title | The effect of CRISPR constructs microinjection on the expression of developmental genes in Rag1 knocked‐out mice embryo |
| title_full | The effect of CRISPR constructs microinjection on the expression of developmental genes in Rag1 knocked‐out mice embryo |
| title_fullStr | The effect of CRISPR constructs microinjection on the expression of developmental genes in Rag1 knocked‐out mice embryo |
| title_full_unstemmed | The effect of CRISPR constructs microinjection on the expression of developmental genes in Rag1 knocked‐out mice embryo |
| title_short | The effect of CRISPR constructs microinjection on the expression of developmental genes in Rag1 knocked‐out mice embryo |
| title_sort | effect of crispr constructs microinjection on the expression of developmental genes in rag1 knocked out mice embryo |
| topic | CRISPR constructs developmental genes microinjection Rag1 knocked‐out embryos |
| url | https://doi.org/10.1002/vms3.380 |
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