Anti-bacterial activity of recombinant human β-defensin-3 secreted in the milk of transgenic goats produced by somatic cell nuclear transfer.

The present study was conducted to determine whether recombinant human β-defensin-3 (rHBD3) in the milk of transgenic goats has an anti-bacterial activity against Escherichia coli (E. coli), Staphylococcus aureus (S. aureus) and Streptococcus agalactiae (S. agalactiae) that could cause mastitis. A HBD3 mammary-specific expression vector was transfected by electroporation into goat fetal fibroblasts which were used to produce fourteen healthy transgenic goats by somatic cell nuclear transfer. The expression level of rHBD3 in the milk of the six transgenic goats ranged from 98 to 121 µg/ml at 15 days of lactation, and was maintained at 90-111 µg/ml during the following 2 months. Milk samples from transgenic goats showed an obvious inhibitory activity against E. coli, S. aureus and S. agalactiae in vitro. The minimal inhibitory concentrations of rHBD3 in milk against E. coli, S. aureus and S. agalactiae were 9.5-10.5, 21.8-23.0 and 17.3-18.5 µg/mL, respectively, which was similar to those of the HBD3 standard (P>0.05). The in vivo anti-bacterial activities of rHBD3 in milk were examined by intramammary infusion of viable bacterial inoculums. We observed that 9/10 and 8/10 glands of non-transgenic goats infused with S. aureus and E. coli became infected. The mean numbers of viable bacteria went up to 2.9×10(3) and 95.4×10(3) CFU/ml at 48 h after infusion, respectively; the mean somatic cell counts (SCC) in infected glands reached up to 260.4×10(5) and 622.2×10(5) cells/ml, which were significantly higher than the SCC in uninfected goat glands. In contrast, no bacteria was presented in glands of transgenic goats and PBS-infused controls, and the SSC did not significantly change throughout the period. Moreover, the compositions and protein profiles of milk from transgenic and non-transgenic goats were identical. The present study demonstrated that HBD3 were an effective anti-bacterial protein to enhance the mastitis resistance of dairy animals

Targeting Human α-Lactalbumin Gene Insertion into the Goat β-Lactoglobulin Locus by TALEN-Mediated Homologous Recombination

<div><p>Special value of goat milk in human nutrition and well being is associated with medical problems of food allergies which are caused by milk proteins such as β-lactoglobulin (BLG). Here, we employed transcription activator-like effector nuclease (TALEN)-assisted homologous recombination in goat fibroblasts to introduce human α-lactalbumin (hLA) genes into goat BLG locus. TALEN-mediated targeting enabled isolation of colonies with mono- and bi-allelic transgene integration in up to 10.1% and 1.1%, respectively, after selection. Specifically, BLG mRNA levels were gradually decreasing in both mo- and bi-allelic goat mammary epithelial cells (GMECs) while hLA demonstrated expression in GMECs in vitro. Gene-targeted fibroblast cells were efficiently used in somatic cell nuclear transfer, resulting in production of hLA knock-in goats directing down-regulated BLG expression and abundant hLA secretion in animal milk. Our findings provide valuable background for animal milk optimization and expedited development for agriculture and biomedicine.</p></div

Human α-lactalbumin gene expression in GMECs in vitro.

<p>(A) Shematic overview dipicting position of primers Bc-F/Bc-R and Hc-F/Hc-R on BLG and hLA exons respectively. Blue boxes, exons of BLG or hLA; Level arrow lines, primers used for RT-PCR. (B) Reverse-transcription PCR analysis of partial BLG (i), hLA (ii) and GAPDH (iii) mRNAs in mono- and bi-allelic BLG-targeted cell clones. NC: non-transfected cells; m1, m2, m3: mono-allelic BLG-targeted clones; b1, b2, b3: bi-allelic BLG-targeted cell clones. (C) qPCR analysis of BLG and hLA expression. WT: non-transfected cells; m1, m2, m3: mono-allelic BLG-targeted clones; b1, b2, b3: bi-allelic BLG-targeted cell clones. The results were derived from at least three different experiments, P<0.05. (D) Western blot analysis for hLA in the supernatants of transfected GMECs. Lane WT, supernatant of non-transfected cells; Lanes 1–2, supernatant of bi-allelic and mono-allelic BLG-targeted cell clones.</p

Goat gene-targeted cloned embryos developed in vitro.

<p>(A) The goat reconstructed embryos cultured in vitro. (B) Seven nuclear cloned embryos deriving from different clones were used for individual-embryo PCR assays to rule out mixed colonies that containing non-targeted cells. Lane 1, non-transgene cloning embryo used for negative control; Lane 2, gene-targeted cell colony used for positive control. Lanes 3–9, reconstructed embros. (C) The gene-targeted goats postpartum (born may 2014).</p

Efficiency of gene targeting in goat somatic cells using TALENs.

<p>Efficiency of gene targeting in goat somatic cells using TALENs.</p

Analysis of knock-in goats.

<p>(A) Junction PCR analysis. Lane WT, wild-type goat; Lanes 1–6, transgenic goats. (B) Nucleotide sequence analysis of the junctions between the endogenous and exogenous DNAs corresponding to HR events. The 1,800-bp and 2,300-bp PCR products specific for the left- and right- hand junctions are indicated by the peak figure below respectively. (C) Southern blot analysis. Lane WT, wild-type goat; Lane 01, bi-allelic targeted goat; Lanes 02–06, mono-allelic targeted goats. (D) Analysis of milk from mono-allelic BLG-targeted goats. (top) Analysis of whey proteins by coomassie blue staining after separation by 15% SDS/PAGE. Equal amounts of milk samples were loaded onto each lane of the gel; (bottom) Western blot analysis of hLA expression. PC, human milk as positive control; WT, wild-type goat; Lanes 02–06, mono-allelic targeted goats.</p

Targeting BLG in goat fibroblasts.

<p>(A) Schematic overview depicting the targeting strategy for the BLG locus. Blue boxes, exons of BLG; Diagonal lines above, TALEN1/2 pair binding sites; Level arrow lines, primers used for PCR; The predicted size of southern hybridization bands with BamHI digestion, for both the endogenous BLG locus and the BLG targeted locus, is indicated. (B) PCR-based measurements of TALEN-driven exogenous gene integration into the BLG locus in goat fibroblasts. Cells were right untransfected (lane 4, for negative control) or were transfected with an expression cassette for TALENs that induce a DSB at exon1 of BLG locus (lane 3), and donor plasmids carrying a foreign gene flanked by two homology arms, in the absence (lane 2) and presence (lane 1) of the TALEN. (C) 3’ Junction PCR results from cell lysis templates. TALEN-mediated transgene insertion yielded 2,300-bp PCR products using primers B31 and B32, which were specific to the neo gene and the BLG locus, respectively. (D) 5’ Junction PCR analysis performed on genomic DNA of 3’ junction PCR-positive colonies using primers B51 and B52 to amplify the 1,800-bp left-hand junction between the endogenous BLG locus and the exogenous hLA.</p