Superovulation, artificial insemination, intradermic injection, and blood collection were performed at the experimental station of the China Agricultural University. The study was carried out in strict accordance with the protocol approved by the Animal Welfare Committee of China Agricultural University (Permit Number: XK662).
Production of Capra hircusin goats over-expressing TLR2
Total RNA was extracted from goat spleens using an RNA kit (OMEGABio-Tek, Doraville, GA, U.S.). cDNA was synthesized using M-MLV reverse transcriptase (Promega, Madison, WI, U.S.) in accordance with the manufacturer’s instructions and following the Capra hircus TLR2 gene sequence (GenBank: GU984768.1). The eukaryotic expression vector pN1 (Plasmid12193; Addgene, Cambridge, MA, U.S.) was used as a backbone for the transformation. It was generated by removal of the EGFP gene from pEGFP-N1. The construct sub-cloned from pIRES2-EGFP (Plasmid12193; Addgene, Cambridge, MA, U.S.) contained an IRES-EGFP fragment connected to pN1 after introduction of a LoxP sequence on either side. This expression vector is called p3S-LoxP. Both the Capra hircus TLR2 sequence and p3S-LoxP were digested before ligation. The new expression vector is here called p3S-LoxP-TLR2.
Healthy laso-shan dairy goats were put into synchronized estrus using CIDR (Pharmacia & UpjohnCompany, Rydalmere, Australia). Ova were collected from donors using superovulation. Zygotes were generated using in vitro fertilization. The zygotes were microinjected with linearized p3S-LoxP-TLR2 solution at concentrations of 5 ng/μL and 10 ng/μL in volumes of 5 pL. They were then transferred into the recipients’ oviducts. DNA was extracted from the ear tissue of each lamb at birth. To identify transgenic individuals, the following PCR primers were used: F: 5′- TCC AAA ATG TCG TAA CAA CTC CG - 3′; R: 5′ - AAA AAG AGA TGT TTC CCC AAG TGT T - 3′. The upstream primer was based on the CMV region and the downstream primer was based on foreign TLR2. For Southern blotting analysis, the PCR product of TLR2 was digested with Nhe I and Hind III (NEB, Beverly, MA, U.S.) and labelled with DIG (12647521;Roche Diagnostics, Mannheim, Germany) for use as a probe. Ear tissue section slides were paraffin-embedded and prepared for immunohistochemical analysis and TLR2 expression analysis. Anti-goat TLR2-FITC was used (ab59711; Abcam, Cambridge, U.K.).
Testing of physiological and biochemical parameters
The blood biochemical parameters of transgenic and control goats were assessed at 120 days. Peripheral blood was collected and both blood cells serum biochemical parameters were assessed. Factors examined here included the white blood cell count (WBC), red blood cell count (RBC), and hemoglobin (HGB), hematocrit (HCT), serum total protein (TP), albumin (Alb), globulin (Glo), alanine aminotransferase (ALT), aspartate aminotransferase (AST), C-reactive protein (CRP), glucose (Glu), blood urea nitrogen (BUN), and triglyceride (TG) levels.
In vitro Pam3CSK4-challenge
Mononuclear cells were isolated from the peripheral blood of 3 transgenic goats and 3 wild goats by density gradient centrifugation. Goat lymphocyte separation medium (TBD, Tianjin, China) was used. Cells were cultured in RPMI 1640 medium (Gibco, Grand Island, NY, U.S.) supplemented with 10% FBS (Gibco, Grand Island, NY, USA). For differential adhesion cultures, the medium was replaced once every 12 hours. During TLR2 receptor agonist experiments, monocytes and macrophages were cultured for 48 hours and stimulated at different times with 1 μg/mL Pam3CSK4 (InvivoGen, San Diego, CA, U.S.). Cell culture supernatants were collected, and the concentrations of IFN-γ, IL-10, IL-6, and LZM were measured using enzyme-linked immunosorbent assay (ELISA) kits. NO and MDA content were detected using a commercial kit (Jiancheng, Nanjing, China). All experimental operations were performed according to kit instructions. Total RNA was extracted from monocyte-macrophages. Real-time PCR was used to evaluate TLR2 expression. TLR2- and β-actin-specific primers were designed (TLR2 F: 5′ - TGC TGT GCC CTC TTC CTG TT - 3′, R: 5′- GGG ACG AAG TCT CGC TTA TGA A - 3′; β-actin F: 5′ - AGA TGT GGA TCA GCA AGC AG - 3′, R: 5′ - CCA ATC TCA TCT CGT TTT CTG - 3′). Relative expression was determined using the comparative 2-△△CT method.
In vivo Pam3CSK4-challenge
Live goats were intradermally injected with 1mg Pam3CSK4 in the ear. Ear tissues were collected at 0.5 hours and 8 hours after Pam3CSK4 stimulation. Samples were fixed with 4% paraformaldehyde in addition to the routine haematoxylin and eosin staining.
Individual experiments were repeated three times. All data were subjected to analysis of variance using the GLM procedures of the statistical analysis system (SAS Institute, U.S.). All data are expressed as mean ± SEM. Differences were considered significant at P< 0.05.