Generation of Calpain-3 knock-out porcine embryos by CRISPR-Cas9 electroporation and intracytoplasmic microinjection of oocytes before insemination

Navarro-Serna, Sergio; Dehesa-Etxebeste, Martxel; Piñeiro-Silva, Celia; Romar, Raquel; Lopes, Jordana S.; Munáin, Adolfo López de; Gadea, J.

doi:10.1016/j.theriogenology.2022.04.012

Cited by 6 publications

(2 citation statements)

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“…No differences were found between the control, electroporated, and lipofected 5% groups, suggesting that the lower concentration had no toxic effect. The electroporated group had a higher cleavage rate than the control group, as determined previously, probably because the oocytes were activated by the electric pulses inducing an influx of calcium from the pulsing medium [18]. In previous studies concerning the electroporation of porcine oocytes, we analyzed the IVF procedure, showing that some of the oocytes presented one pronucleus, although the majority of them, and in a similar rate to the control, presented two pronuclei (male and female; data not shown).…”

Section: Discussionsupporting

confidence: 68%

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Production of Genetically Modified Porcine Embryos via Lipofection of Zona-Pellucida-Intact Oocytes Using the CRISPR/Cas9 System

Piñeiro-Silva

Navarro-Serna

Belda-Pérez

et al. 2023

Animals

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The generation of genetically modified pigs has an important impact thanks its applications in basic research, biomedicine, and meat production. Cloning was the first technique used for this production, although easier and cheaper methods were developed, such as the microinjection, electroporation, or lipofection of oocytes and zygotes. In this study, we analyzed the production of genetically modified embryos via lipofection of zona-pellucida-intact oocytes using LipofectamineTM CRISPRMAXTM Cas9 in comparison with the electroporation method. Two factors were evaluated: (i) the increment in the concentration of the lipofectamine–ribonucleoprotein complexes (LRNPC) (5% vs. 10%) and (ii) the concentration of ribonucleoprotein within the complexes (1xRNP vs. 2xRNP). We found that the increment in the concentration of the LRNPC had a detrimental effect on embryo development and a subsequent effect on the number of mutant embryos. The 5% group had a similar mutant blastocyst rate to the electroporation method (5.52% and 6.38%, respectively, p > 0.05). The increment in the concentration of the ribonucleoprotein inside the complexes had no effect on the blastocyst rate and mutation rate, with the mutant blastocyst rate being similar in both the 1xRNP and 2xRNP lipofection groups and the electroporation group (1.75%, 3.60%, and 3.57%, respectively, p > 0.05). Here, we showed that it is possible to produce knock-out embryos via lipofection of zona-pellucida-intact porcine oocytes with similar efficiencies as with electroporation, although more optimization is needed, mainly in terms of the use of more efficient vesicles for encapsulation with different compositions.

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Section: Discussionsupporting

confidence: 68%

“…However, SCNT and microinjection are difficult to perform, requiring specific equipment and trained personnel. While the electroporation technique is the easiest one to perform, an electroporator is needed, and the efficiency of generating KO porcine embryos is equivalent to that achieved via microinjection [18].…”

Section: Introductionmentioning

confidence: 99%