Anti-Bacterial Activity of Recombinant Human β-Defensin-3 Secreted in the Milk of Transgenic Goats Produced by Somatic Cell Nuclear Transfer

Abstract

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 2.9×103 and 95.4×103 CFU/ml at 48 h after infusion, respectively; the mean somatic cell counts (SCC) in infected glands reached up to 260.4×105 and 622.2×105 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.

Goat Uterine DBA+ Leukocytes Differentiation and Cytokines Expression Respond Differently to Cloned versus Fertilized Embryos

“…In the present study, IVF embryos were used as a control for the identification of specific responses of the uterine DBA + leukocytes derived from non-pregnant endometrium to SCNT embryos. The IVF protocol used in this study had been previously shown to produce fetuses that are not different from in vivo -derived fetuses in growth and endocrine parameters [ 18 ]. DBA lectin reactivity has been widely used for histological recognition and isolation of uterine NK cells in pregnant mice [ 14 , 15 ].…”

A promoter trap vector for knocking out bovine myostatin gene with high targeting efficiency

“…Since the targeting of COL1A1 in sheep (McCreath et al, 2000), somatic cell nuclear transfer technology has provided an alternative means of cell-mediated transgenesis to circumvent the requirement for embryonic stem cells to generate gene-targeted animals such as sheep (Denning et al, 2001), cattle (Kuroiwa et al, 2009), goat (Liu et al, 2013), and pigs (Dai et al, 2002;Lai et al, 2002). It is technically challenging to produce gene-targeted cell lines as donors with sufficient genetic stability for somatic cell nuclear transfer.…”

Generation of β‐lactoglobulin‐modified transgenic goats by homologous recombination

“…The SCNT procedure was conducted as described previously . Briefly, the polar body and metaphase plate were sucked out, and a single round donor cell was injected into the perivitelline space of the enucleated oocytes.…”