Efficient production of GGTA1 knockout porcine embryos using a modified handmade cloning (HMC) method

Kumbha, Ramesh; Hosny, Nora; Matson, Anders W.; Steinhoff, Magie; Hering, Bernhard J.; Burlak, Christopher

doi:10.1016/j.rvsc.2019.10.021

Cited by 3 publications

(2 citation statements)

References 60 publications

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“…Somatic cells, such as fetal fibroblasts, are transformed with plasmids encoding for the Cas9 and the sgRNAs, along with a reporter gene and/or an antibiotic resistance gene; allowing the screening and/or selection of the modified cells (Ren et al, 2019). Once the edited cells are obtained, they are used to generate founder pigs, which will present a predictable genotype avoiding mosaicism (Chen et al, 2015;Wang K. et al, 2015;Kumbha et al, 2020). Furthermore, the multi-targeting capacity of the CRISPR-Cas9 system allows to edit many target genes simultaneously, a feature used by Niu et al (2017), to produce porcine retrovirus PERV-free pigs by SCNT, where 62 copies of this retrovirus were edited.…”

Section: Scntmentioning

confidence: 99%

Practical Approaches for Knock-Out Gene Editing in Pigs

et al. 2021

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Pigs are an important resource for meat production and serve as a model for human diseases. Due to their physiological and anatomical similarities to humans, these animals can recapitulate symptoms of human diseases, becoming an effective model for biomedical research. Although, in the past pig have not been widely used partially because of the difficulty in genetic modification; nowadays, with the new revolutionary technology of programmable nucleases, and fundamentally of the CRISPR-Cas9 systems, it is possible for the first time to precisely modify the porcine genome as never before. To this purpose, it is necessary to introduce the system into early stage zygotes or to edit cells followed by somatic cell nuclear transfer. In this review, several strategies for pig knock-out gene editing, using the CRISPR-Cas9 system, will be summarized, as well as genotyping methods and different delivery techniques to introduce these tools into the embryos. Finally, the best approaches to produce homogeneous, biallelic edited animals will be discussed.

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Section: Scntmentioning

confidence: 99%

Practical Approaches for Knock-Out Gene Editing in Pigs

et al. 2021

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“…However, the most promising method of artificial activation of oocytes is chemical stimulation. For this purpose, there are used ethanol (Fujinami et al, 2004;Báez et al, 2021), calcium ionophore A213187 (Hosseini et al, 2008;Xu et al, 2021), ionomycin (Méo et al, 2008;Jia et al, 2023), puromycin (De Sutter et al, 1992de Castro et al, 2022), strontium chloride (Kim et al, 2014;Fawzy et al, 2018), phorbol ester (Cuthbertson & Cobbold, 1985;Zhao et al, 2014), thimerosal (Kumbha et al, 2020), dehydroleukodine, ionomycin, anisomycin, cycloheximide, 6-dimethylaminopurine (Vichera et al, 2002, alone and in combinations. Addition of those compounds is mostly oriented at mobilization of intracellular calcium by depletion of its reserves, release of intracellular reserves of calcium and promotion of ingress of extracellular calcium ions inside the cell.…”

Section: Introductionmentioning

confidence: 99%

Influence of calcium ionophore on the fertilization of bovine oocytes and their further embryonic development

Kovpak

DERKACH

et al. 2023

Regul. Mech. Biosyst.

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Intracytoplasmic spermatozoid injection (ICSI) is one of the commonest methods used in assisted reproductive technologies in human medicine. However, this procedure has low efficacy for bovines, mainly because of insufficient activation of oocytes after spermatozoid microinjection. One of the most effective methods of activating oocytes is considered to be the use of phosphorus calcium, though the optimal concentration of activator and its effect on pre-implant development of embyo are still open questions. An oocyte-cumulus complex of clinically healthy cows, retrieved from the ovaries during slaughter, matured over 22–24 h in in vitro conditions. Oocytes with visible polar body had been subjected to intracytoplasmic spermatozoid injection (ICSI), and were 15–30 min later activated in the environment with different concentrations of calcium ionophore for 15–20 min and then transferred for further cultivation in a culture medium with sodium pyruvate. The fertilization rate was identified on the second day at the 2–4th stages of cellular embryo, and the quality of obtained embyos was evaluated on day 8. Based on the statistical analysis of the data, we determined that the artificial activation of bovine oocytes using calcium ionphore after intracytoplasmic spermatozoid injection (ICSI) led to statistically significant improvement in conception and ratio of blastocytes obtained to oocytes injected. In the study, we confirmed that addition of 5, 10 and 50 µМ of the agent had the same efficacy on the activation of occytes of bovine cattle. However, it has to be noted that during further cultivation of the obtained zygotes up to the blastocyte stage (day 8), we saw no significant differences in quality of embryos obtained. Therefore, use of calcium ionophore for the activation of bovine oocytes after intracytoplasmic spermatozoid injection is effective, for it promotes increase in fertilization parameters and ratio of blastocytes obtained to oocytes injected, facilitating production of higher numbers of embyos suitable for transplantation or cooling. Our previous conclusions are valuable for increasing the efficacy of methods of intracytoplasmic injection of bovine spermatozoid and its further use for purposes of science and production.

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Recent advances in critical nodes of embryo engineering technology

Ma¹,

Gu²,

Chen³

et al. 2021

Theranostics

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The normal development and maturation of oocytes and sperm, the formation of fertilized ova, the implantation of early embryos, and the growth and development of foetuses are the biological basis of mammalian reproduction. Therefore, research on oocytes has always occupied a very important position in the life sciences and reproductive medicine fields. Various embryo engineering technologies for oocytes, early embryo formation and subsequent developmental stages and different target sites, such as gene editing, intracytoplasmic sperm injection (ICSI), preimplantation genetic diagnosis (PGD), and somatic cell nuclear transfer (SCNT) technologies, have all been established and widely used in industrialization. However, as research continues to deepen and target species become more advanced, embryo engineering technology has also been developing in a more complex and sophisticated direction. At the same time, the success rate also shows a declining trend, resulting in an extension of the research and development cycle and rising costs. By studying the existing embryo engineering technology process, we discovered three critical nodes that have the greatest impact on the development of oocytes and early embryos, namely, oocyte micromanipulation, oocyte electrical activation/reconstructed embryo electrofusion, and the in vitro culture of early embryos. This article mainly demonstrates the efforts made by researchers in the relevant technologies of these three critical nodes from an engineering perspective, analyses the shortcomings of the current technology, and proposes a plan and prospects for the development of embryo engineering technology in the future.

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Efficient production of GGTA1 knockout porcine embryos using a modified handmade cloning (HMC) method

Cited by 3 publications

References 60 publications

Practical Approaches for Knock-Out Gene Editing in Pigs

Practical Approaches for Knock-Out Gene Editing in Pigs

Influence of calcium ionophore on the fertilization of bovine oocytes and their further embryonic development

Recent advances in critical nodes of embryo engineering technology

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