Optimization of lipofection protocols for CRISPR/Cas9 delivery in porcine zona pellucida intact oocytes: A study of coincubation duration and reagent efficacy.

A priority to facilitate the application of lipofection to generate genetically modified porcine embryos and animals will be the use of zona pellucida (ZP)-intact oocytes and zygotes. Recently, our group produced genetically modified embryos by lipofection of ZP-intact oocytes during in vitro fertilization (IVF). This study investigates the effect of two commercial lipofection reagents, Lipofectamine 3000 and Lipofectamine CRISPRMAX, on embryo development and mutation efficiency in ZP-intact porcine oocytes.

Optimizing oocyte electroporation for genetic modification of porcine embryos: Evaluation of the parthenogenetic activation.

In reproductive biology, understanding the effects of novel techniques on early embryo development is of paramount importance. To date, the effects of electrical activation on oocytes prior to in vitro fertilization (IVF) are not well understood. The aim of this study was to investigate the effects of oocyte electroporation prior to IVF on embryo development and to differentiate between true embryos and parthenotes by using a TPCN2 knock-out (KO) male to evaluate the presence of the KO allele in the resulting blastocysts.

Oocyte electroporation prior to in vitro fertilization is an efficient method to generate single, double, and multiple knockout porcine embryos of interest in biomedicine and animal production.

Genetically modified pigs play a critical role in mimicking human diseases, xenotransplantation, and the development of pigs resistant to viral diseases. The use of programmable endonucleases, including the CRISPR/Cas9 system, has revolutionized the generation of genetically modified pigs. This study evaluates the efficiency of electroporation of oocytes prior to fertilization in generating edited gene embryos for different models.

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

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 Lipofectamine CRISPRMAX Cas9 in comparison with the electroporation method.

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

Limb girdle muscular dystrophy type R1 (LGMDR1) is an autosomal recessive myopathy described in humans resulting from a deficiency of calpain-3 protein (CAPN3). This disease lacks effective treatment and an appropriate model, so the generation of KO pigs by CRISPR-Cas9 offers a way to better understand disease ethology and to develop novel therapies. Microinjection is the main method described for gene editing by CRISPR-Cas9 in porcine embryo, but electroporation, which allows handling more embryos faster and easier, has also recently been reported.

Effect of Aphidicolin, a Reversible Inhibitor of Eukaryotic Nuclear DNA Replication, on the Production of Genetically Modified Porcine Embryos by CRISPR/Cas9.

Mosaicism is the most important limitation for one-step gene editing in embryos by CRISPR/Cas9 because cuts and repairs sometimes take place after the first DNA replication of the zygote. To try to minimize the risk of mosaicism, in this study a reversible DNA replication inhibitor was used after the release of CRISPR/Cas9 in the cell. There is no previous information on the use of aphidicolin in porcine embryos, so the reversible inhibition of DNA replication and the effect on embryo development of different concentrations of this drug was first evaluated.