Generating Cloned Goats by Somatic Cell Nuclear Transfer—Molecular Determinants and Application to Transgenics and Biomedicine

Skrzyszowska, M.; Samiec, M.

doi:10.3390/ijms22147490

Cited by 31 publications

(22 citation statements)

References 69 publications

(122 reference statements)

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“…Collectively, the results of the current investigation aimed to devise and optimize intracellular strain-related parameters that turned out to vary depending on different speeds of penetrating the porcine oocytes with the use of robotic (i.e., motorized) micro-manipulators might contribute to improvement of the efficiency of a variety of modern assisted reproductive technologies (ARTs). Taking the above-mentioned findings into consideration, the augmented efficiencies of microsurgical in vitro fertilization by ICSI and cloning by somatic cell nuclear transfer (SCNT) will be of especially great importance not only in pigs but also in other mammalian species [25][26][27][28][29][30][31].…”

Section: Discussionmentioning

confidence: 99%

Oocyte Penetration Speed Optimization Based on Intracellular Strain

et al. 2022

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Oocyte penetration is an essential step for many biological technologies, such as animal cloning, embryo microinjection, and intracytoplasmic sperm injection (ICSI). Although the success rate of robotic cell penetration is very high now, the development potential of oocytes after penetration has not been significantly improved compared with manual operation. In this paper, we optimized the oocyte penetration speed based on the intracellular strain. We firstly analyzed the intracellular strain at different penetration speeds and performed the penetration experiments on porcine oocytes. Secondly, we studied the cell development potential after penetration at different penetration speeds. The statistical results showed that the percentage of large intracellular strain decreased by 80% and the maximum and average intracellular strain decreased by 25–38% at the penetration speed of 50 μm/s compared to at 10 μm/s. Experiment results showed that the cleavage rates of the oocytes after penetration increased from 65.56% to 86.36%, as the penetration speed increased from 10 to 50 μm/s. Finally, we verified the gene expression of oocytes after penetration at different speeds. The experimental results showed that the totipotency and antiapoptotic genes of oocytes were significantly higher after penetration at the speed of 50 μm/s, which verified the effectiveness of the optimization method at the gene level.

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Section: Discussionmentioning

confidence: 99%

Oocyte Penetration Speed Optimization Based on Intracellular Strain

et al. 2022

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“…Recently, the clustered regularly interspaced short palindromic repeats (CRISPRs)/CRISPR-associated (Cas) 9 system was developed to edit specific genes with high efficiency [ 16 , 17 ]. Using this technique, numerous genome-edited animals from different species have been generated for biomedical modeling [ 18 , 19 , 20 , 21 ], human disease modeling [ 22 , 23 , 24 ], and xenotransplantation [ 25 , 26 , 27 ]. In fact, apoE knockout (KO) dogs [ 28 ] and myostatin KO dogs [ 29 ] were successfully produced by microinjecting CRISPR/Cas9 into zygotes.…”

Section: Introductionmentioning

confidence: 99%

Generation of a Dystrophin Mutant in Dog by Nuclear Transfer Using CRISPR/Cas9-Mediated Somatic Cells: A Preliminary Study

Chung

Kim

et al. 2022

IJMS

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Dystrophinopathy is caused by mutations in the dystrophin gene, which lead to progressive muscle degeneration, necrosis, and finally, death. Recently, golden retrievers have been suggested as a useful animal model for studying human dystrophinopathy, but the model has limitations due to difficulty in maintaining the genetic background using conventional breeding. In this study, we successfully generated a dystrophin mutant dog using the CRISPR/Cas9 system and somatic cell nuclear transfer. The dystrophin mutant dog displayed phenotypes such as elevated serum creatine kinase, dystrophin deficiency, skeletal muscle defects, an abnormal electrocardiogram, and avoidance of ambulation. These results indicate that donor cells with CRISPR/Cas9 for a specific gene combined with the somatic cell nuclear transfer technique can efficiently produce a dystrophin mutant dog, which will help in the successful development of gene therapy drugs for dogs and humans.

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“…Somatic cell nuclear transfer (SCNT) is currently the most widely used to produce large‐ and medium‐sized transgenic animals (Czernik et al., 2019; Skrzyszowska & Samiec, 2021; Yin et al., 2019). Most of the donor cells for SCNT are long‐term cultured in vitro and have defects such as reduced cell growth potential and ageing quickly, which affected the fusion and development of the reconstructed embryos (Ao et al., 2020; Brochard & Beaujean, 2021; Zhou et al., 2020).…”

Section: Introductionmentioning

confidence: 99%

Developmental analysis of reconstructed embryos of second‐generation cloned transgenic goats

Song

Lü

Cheng

et al. 2022

Reprod Domestic Animals

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To improve the efficiency of the production of transgenic cloned goats by somatic cell nuclear transfer (SCNT), the development of reconstructed embryos of firstgeneration (G1) and second-generation (G2) cloned transgenic goats was compared and analysed. Primary transgenic foetal fibroblasts were used as donor cells for G1 somatic cell nuclear transfer (SCNT). When the G1 transgenic embryos developed to 35 days in the recipient goats, transgenic foetal fibroblasts were isolated from them and used as donor cells for the G2 clone. In the G1 clones, the average fusion rate of reconstructed embryos was 73.62 ± 2.9%, the average development rate(2-4 cells) was 33.96 ± 2.36%, and the pregnancy rate of transplant recipients was 31.91%. In the G2 clones, the average fusion rate of the reconstructed embryos was 90.29 ± 2.03%, the average development rate was 66.46 ± 3.30%, and the pregnancy rate was 58.14%. These results indicate that in the G2 clones, the fusion rate of eggs, the development rate of reconstructed embryos and the pregnancy rate of transplant recipients were significantly higher than those of G1 clones. We believe these results will lay a solid foundation for the efficient production of transgenic cloned animals in the future.

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Generating Cloned Goats by Somatic Cell Nuclear Transfer—Molecular Determinants and Application to Transgenics and Biomedicine

Cited by 31 publications

References 69 publications

Oocyte Penetration Speed Optimization Based on Intracellular Strain

Oocyte Penetration Speed Optimization Based on Intracellular Strain

Generation of a Dystrophin Mutant in Dog by Nuclear Transfer Using CRISPR/Cas9-Mediated Somatic Cells: A Preliminary Study

Developmental analysis of reconstructed embryos of second‐generation cloned transgenic goats

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