Efficient delivery of a large-size Cas9-EGFP vector in porcine fetal fibroblasts using a Lonza 4D-Nucleofector system.

Background: Porcine fetal fibroblasts (PFFs) are important donor cells for generating genetically modified pigs, but the transfection efficiencies of PFFs are often unsatisfactory especially when large-size vectors are to be delivered. In this study, we aimed to optimize the transfection conditions for delivery of a large-size vector in PFFs using Lonza 4D-Nucleofector™ vessels and strips.

Methods: We firstly delivered a 13 kb Cas9-EGFP and a 3.

Highly Efficient A-to-G Editing in PFFs via Multiple ABEs.

Cytosine base editors (CBEs) and adenine base editors (ABEs) are recently developed CRISPR-mediated genome-editing tools that do not introduce double-strand breaks. In this study, five ABEs, ABE7.10, ABEmax, NG-ABEmax, ABE8e and NG-ABE8e, were used to generate A-to-G (T-to-C) conversions in five genome loci in porcine fetal fibroblasts (PFFs).

Efficient Simultaneous Introduction of Premature Stop Codons in Three Tumor Suppressor Genes in PFFs via a Cytosine Base Editor.

Base editing is an efficient and precise gene-editing technique, by which a single base can be changed without introducing double-strand breaks, and it is currently widely used in studies of various species. In this study, we used hA3A-BE3-Y130F to simultaneously introduce premature stop codons (TAG, TGA, and TAA) into three tumor suppressor genes, TP53, PTEN, and APC, in large white porcine fetal fibroblasts (PFFs). Among the isolated 290 single-cell colonies, 232 (80%) had premature stop codons in all the three genes.

gene disruption causes severe limb deformities in pigs.

In an attempt to generate g.A746G substitution in the gene, we unexpectedly obtained homozygous knockout piglets ( ) and heterogeneous knockout piglets with one copy of the A746G mutation ( ) via CRISPR/Cas9 editing. Polymerase chain reaction (PCR) and sequencing revealed complex genomic rearrangements in the target region.

Phosphorylation of histone H3 on Ser-10 by Aurora B is essential for chromosome condensation in porcine embryos during the first mitotic division.

Phosphorylation of histone H3 on Ser-10 (H3S10ph) is involved in regulating mitotic chromosome condensation and decondensation, which plays an important regulatory role during mitotic cell cycle progression in mammalian cells. However, whether H3S10ph plays a similar role in early porcine embryos during the first mitotic division remains uncertain. In this study, the subcellular localization and possible roles of H3S10ph were evaluated in the first mitotic cell cycle progression of porcine embryos using western blot, indirect immunofluorescence and barasertib (H3S10ph upstream regulator Aurora-B inhibitor) treatments.

Plk1 inhibition leads to a failure of mitotic division during the first mitotic division in pig embryos.

Purpose: This study was conducted to examine the dynamic distribution of polo-like 1 kinase (Plk1) and the possible role it plays in first mitotic division during early porcine embryo development.

Methods: Indirect immunofluorescence and confocal microscopy imaging techniques combined with western blot analyses were used to study the dynamic expression and subcellular localization of Plk1 protein in pig parthenogenetic embryos. Finally, a selective Plk1 inhibitor, GSK461364, was used to evaluate the potential role of Plk1 during this special stage.