Generation of Marker-Free pbd-2 Knock-in Pigs Using the CRISPR/Cas9 and Cre/loxP Systems

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.

Keywords: CRISPR/Cas9; antimicrobial peptide; disease-resistant animals; porcine β-defensin 2; transgenic pigs.

Figure 1

Site-specific pbd-2 knock-in at the porcine Rosa26 (pRosa26) locus. (A) Map of the donor plasmid pcCAG-R26-PBD2-T2A-PBD2 for the site-specific pbd-2 knock-in. (B) Scheme for marker-free targeted pbd-2 integration in porcine fetal fibroblasts (PFFs) via CRISPR/Cas9-mediated homology-directed repair. The floxed selectable marker gene (SMG) was removed after treatment of Cre recombinase. (C) Relative copy number of the SMG neoR in wild-type (WT) PFFs, transgenic (TG) PFFs and Cre-recombinase-treated TG PFFs. (D) The bactericidal activities of cell culture supernatants of WT PFFs and TG PFFs on Actinobacillus pleuropneumoniae and Streptococcus suis quantified by bacterial counting. Data are presented as mean ± SD and are plotted from three independent experiments. * p < 0.05, **** p < 0.0001, unpaired one tailed Student′ s t-test.

Figure 2

Genotyping of cloned piglets. (A) Physical appearance of cloned piglets. (B) PCR analysis to identify marker-free site-specific pbd-2 knock-in pigs. PBD-2: Amplification for the dual pbd-2 gene using primers vTG-F and vTG-R; NeoR: Amplification for the SMG neoR; Off-target: Amplification for the fragment which represents on-target insertion of the transgene using primers OT-F and OT-R; β-actin: Amplification for β-actin; Lane 220–227: Numbers for pigs; BC: Blank control; PC: Positive control. (C) Southern blot analysis for the identification of TG pigs. Genomic DNA from porcine ear fibroblasts (PEFs) was extracted and digested with XhoI, followed by Southern blot analysis using a digoxigenin-labeled pbd-2-specific probe. M: Molecular mass marker; Lane 220–230: Numbers for pigs; PC: Positive control.

Figure 3

Off-target analysis. (A) Predicted off-target sites. Lower case letters represent mismatched bases, while underlined letters represent the 3′ PAM of the target sequence. (B) Sanger sequencing results of the PCR products of potential off-target sites.

Figure 4

Transcriptional and translational analysis of pbd-2 in cloned pigs. (A) The mRNA expression level of pbd-2 in different organs. Total RNA in different organs of TG and WT pigs was extracted and then subjected to reverse transcription, followed by RT-qPCR to determine the mRNA level of pbd-2, GAPDH was used as an internal reference gene. Data are presented as mean ± SD and are plotted from three independent experiments. ** p < 0.01, *** p < 0.001, **** p < 0.0001, n = 3, unpaired one tailed Student′ s t-test. (B) Immunofluorescent analysis of the expression of porcine β-defensin 2 (PBD-2) using a 40× objective and a 100 ms exposure. The expression of PBD-2 in PEFs from TG pigs was detected by immunofluorescent analysis using mouse monoclonal anti-PBD2 antibody followed by FITC-labeled goat anti-mouse IgG. Cell nuclei were stained with 4′,6-diamidino-2-phenylindole, the PEFs from WT pigs served as a negative control. Scale bar = 100 μm. (C) Immunohistochemical analysis of the expression of PBD-2 at a magnification of 200×, with an exposure time of 50 ms. Expression of PBD-2 in heart, liver, spleen, lung, kidney and brain tissues of WT and TG pigs was determined by immunohistochemistry using mouse monoclonal 2A peptide antibody. Brown color represents the detected 2A peptide, which is co-expressed with PBD-2. Scale bars = 100 μm.

Figure 5

Bactericidal activity of cell culture supernatant. The cell culture supernatant of PEFs from TG pigs was incubated with A. pleuropneumoniae and S. suis for 1 h and the surviving bacteria were then plated on agar for counting. Cell culture supernatant of PEFs from WT pigs was used as a negative control. Data are presented as mean ± SD and are plotted from three independent experiments. * p < 0.05, ** p < 0.01, unpaired one tailed Student′ s t-test.