Secretion of IFN-γ by Transgenic Mammary Epithelial Cells in vitro Reduced Mastitis Infection Risk in Goats

Abstract

Mastitis results in great economic loss to the dairy goat industry. Many approaches have attempted to decrease the morbidity associated with this disease, and among these, transgenic strategy have been recognized as a potential approach. A previous mammalian study reports that interferon-gamma (IFN-γ) has potential anti-bacterial bioactivity against infection in vitro; however, its capacity in vivo is ambiguous. In this study, we initially constructed targeting and homologous recombination vectors (containing the IFN-γ gene) and then transferred the vectors into goat mammary gland epithelial cells (GMECs). Enzyme digestion and sequencing analysis indicated that the vectors used in this study were built correctly. Subsequently, monoclonal cells were selected using puromycin and the polymerase chain reaction (PCR) test indicated that IFN-γ was correctly inserted downstream of the casein promoter. Monoclonal cells were then assessed for reducible expression, and reverse transcriptase-PCR (RT-PCR) and Western blot tests confirmed that monoclonal cells could express IFN-γ. Finally, anti-bacterial capacity was evaluated using bacterial counts and flow cytometry analysis. Decreased bacterial counts and cell apoptosis rates in transgenic GMECs demonstrated that the secretion of IFN-γ could inhibit bacterial proliferation. Therefore, IFN-γ gene transfection in goat mammary epithelial cells could inhibit bacterial proliferation and reduce the risk of mammary gland infection in goats.

Keywords: IFN-γ; anti-mastitis activity; gene-editing; infection risk; mastitis.

Figure 1

The entire homologous integration scheme of this research. In the strategy, one target site was chosen in the 2nd exon of the casein (CSN2) gene, upstream of the signal peptide of the gene. The other site was located upstream of the 8th exon. The homologous arm (long arm: LA) of the recombination vector was designed containing the CSN2 signal peptide and used for guiding interferon-gamma (IFN-γ) secretion.

Figure 2

Construction of gene-targeted and homologous integration vectors. (A) Location of single guide RNAs (sgRNAs) that were used in this study. (B) Sequencing of the target vector; the sgRNAs were inserted into the Lenti-CRISPR V2 vector followed by sequencing. (C) Digestion efficiency analysis of sgRNAs. (D–G) Homologous recombination vector construction process. (D) The homologous arm was amplified and assessed by polymerase chain reaction (PCR) assay. The bands in line 1 and lane 2 independently represent the long and the short arm length. (E) Interferon-gamma (IFN-γ) was synthesized and inserted downstream of the long arm by overlap PCR, which was about 1,358 bp in length. (F) Long arm inserting assay, the vector was double digested by Spe I and EcoR I enzymes. Two bands in lane 1 and 2 showed that the long arm with IFN-γ was completely inserted into the homologous vector. (G) Enzyme identification of the entire combination vector. The short arm was digested from the recombination vector by Kpn I.

Figure 3

Transgenic monoclonal goat mammary gland epithelial cells (GMECs) screening and evaluation. (A) Lentivirus plasmids were packaged in 293T cells. Homologous and target vectors were all packaged in 293T cells. Expression of green fluorescent protein (GFP) in 293T cells confirmed that the infection rate of homologous vectors was sufficient for the virus concentration. (B) The monoclonal GMECs that were obtained after puromycin selection. (C,D) Integrated interferon-gamma (IFN-γ) expression cassettes were evaluated by PCR assays. Inserted site detection indicated the expression cassettes were correctly integrated into the genome of GMECs. Middle lane (D) in the figure is the KY2 PCR product, the right lane (D) is KY1 PCR product. (E) Detection of IFN-γ mRNA in the monoclonal cells by RT-PCR after inducible expression. (F) Western blot analysis of inducible IFN-γ. Analysis of IFN-γ indicated that the gene-edited GMECs could secrete IFN-γ protein as expected.

Figure 4

Anti-bacterial capacity of transgenic monoclonal goat mammary gland epithelial cells (GMECs) that expressed and secreted interferon-gamma (IFN-γ) protein. (A–C) Bacterial challenge of transgenic GMECs was performed to assess the anti-bacterial capacity of IFN-γ. Transgenic GMECs showed much stronger Staphylococcus and Escherichia coli resistant activity than the non-transgenic ones (P < 0.01), but no obvious differences in anti-Streptococcus effects. (D,E) Survival rate of GMECs in different bacteria-treated cells at 6 h; transgenic GMECs obviously increased the survival rate over non-transgenic ones (P < 0.05). *p < 0.05, **p < 0.01.