Abstract
Porcine β-defensin 2 (PBD-2), expressed by different tissues of pigs, is a multifunctional cationic peptide with antimicrobial, immunomodulatory and growth-promoting abilities. As the latest generation of genome-editing tool, CRISPR/Cas9 system makes it possible to enhance the expression of PBD-2 in pigs by site-specific knock-in of pbd-2 gene into the pig genome. In this study, we aimed to generate marker-free pbd-2 knock-in pigs using the CRISPR/Cas9 and Cre/loxP systems. Two copies of pbd-2 gene linked by a T2A sequence were inserted into the porcine Rosa26 locus through CRISPR/Cas9-mediated homology-directed repair. The floxed selectable marker gene neoR, used for G418 screening of positive cell clones, was removed by cell-penetrating Cre recombinase with a recombination efficiency of 48.3%. Cloned piglets were produced via somatic cell nuclear transfer and correct insertion of pbd-2 genes was confirmed by PCR and Southern blot. Immunohistochemistry and immunofluorescence analyses indicated that expression levels of PBD-2 in different tissues of transgenic (TG) piglets were significantly higher than those of their wild-type (WT) littermates. Bactericidal assays demonstrated that there was a significant increase in the antimicrobial properties of the cell culture supernatants of porcine ear fibroblasts from the TG pigs in comparison to those from the WT pigs. Altogether, our study improved the protein expression level of PBD-2 in pigs by site-specific integration of pbd-2 into the pig genome, which not only provided an effective pig model to study the anti-infection mechanisms of PBD-2 but also a promising genetic material for the breeding of disease-resistant pigs.
Highlights
Different breeding techniques have been utilized to improve animal production traits including growth rate, milk yield and disease-resistance
Streptococcus suis strain SC19 was cultured in tryptic soy broth (TSB; BD, Franklin Lakes, NJ, USA) with 5% newborn calf serum (NBCS; TIANHANG, Huzhou, China) and on tryptic soy agar (TSA; BD) with 5% NBCS
A. pleuropneumoniae strain 4074 was grown in TSB with 5% NBCS and 10 μg/mL of nicotinamide adenine dinucleotide
Summary
Different breeding techniques have been utilized to improve animal production traits including growth rate, milk yield and disease-resistance. With the rapid development of biotechnologies, transgenic (TG) techniques have been gradually applied in animal breeding. There are three major gene-editing tools used, named as zinc finger nuclease, transcription activator-like effector nuclease and CRISPR/Cas9 [1,2,3]. Events of using gene-editing tools to improve quantitative traits and welfare of animals, and to eliminate allergens in livestock products, have been well described. Eggs with low allergenicity were produced by gene disruptions of OVA and OVM genes in hens [8], while β-lactoglobulin-free goat milk was obtained by removing BLG gene in goats [9]
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