• Animal Bioscience
• /
• 제35권1호
• /
• pp.126-137
• /
• 2022

Objective: Efficient gene editing technology is critical for successful knock-in in domestic animals. RAD51 recombinase (RAD51) gene plays an important role in strand invasion during homologous recombination (HR) in mammals, and is regulated by checkpoint kinase 1 (CHK1) and CHK2 genes, which are upstream elements of RAD51 recombinase (RAD51). In addition, mismatch repair (MMR) system is inextricably linked to HR-related pathways and regulates HR via heteroduplex rejection. Thus, the aim of this study was to investigate whether clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9)-mediated knock-in efficiency of human lactoferrin (hLF) knock-in vector in the bovine β-casein gene locus can be increased by suppressing DNA MMR-related genes (MSH2, MSH3, MSH6, MLH1, and PMS2) and overexpressing DNA double-strand break (DSB) repair-related genes (RAD51, CHK1, CHK2). Methods: Bovine mammary epithelial (MAC-T) cells were transfected with a knock-in vector, RAD51, CHK1, or CHK2 overexpression vector and CRISPR/sgRNA expression vector to target the bovine β-casein gene locus, followed by treatment of the cells with CdCl₂ for 24 hours. After 3 days of CdCl₂ treatment, the knock-in efficiency was confirmed by polymerase chain reaction (PCR). The mRNA expression levels of DNA MMR-related and DNA DSB repair-related genes were assessed by quantitative real-time PCR (RT-qPCR). Results: Treatment with CdCl₂ decreased the mRNA expression of RAD51 and MMRrelated genes but did not increase the knock-in efficiency in MAC-T cells. Also, the overexpression of DNA DSB repair-related genes in MAC-T cells did not significantly affect the mRNA expression of MMR-related genes and failed to increase the knock-in efficiency. Conclusion: Treatment with CdCl₂ inhibited the mRNA levels of RAD51 and DNA MMR-related genes in MAC-T cells. However, the function of MMR pathway in relation to HR may differ in various cell types or species.

• Journal of Microbiology and Biotechnology
• /
• 제30권10호
• /
• pp.1458-1466
• /
• 2020

Oligomeric proanthocyanidins (OPCs), classified as condensed tannins, have significant antioxidation, anti-inflammation and anti-cancer effects. This study was performed to investigate the anti-inflammatory effects of OPCs and the mechanism underlying these effects in lipopolysaccharide (LPS)-stimulated bovine mammary epithelial cells (MAC-T). Real-time PCR and ELISA assays indicated that OPC treatment at 1, 3 and 5 ㎍/ml significantly reduced the mRNA and protein, respectively, of oxidant indicators cyclooxygenase-2 (COX-2) (p < 0.05) and inducible nitric oxide synthase (iNOS) (p < 0.01) as well as inflammation cytokines interleukin (IL)-6 (p < 0.01), IL-1β (p < 0.01) and tumor necrosis factor-α (TNF-α) (p < 0.05) in LPS-induced MAC-T cells. Moreover, OPCs downregulated LPS-induced phosphorylation of p65 and inhibitor of nuclear factor kappa B (NF-κB) (IκB) in the NF-κB signaling pathway (p < 0.01), and they inhibited p65 translocation from the cytoplasm to the nucleus as revealed by immunofluorescence test and western blot. Additionally, OPCs decreased phosphorylation of p38, extracellular signal regulated kinase and c-jun NH₂-terminal kinase in the MAPK signaling pathway (p < 0.01). In conclusion, the anti-inflammatory and antioxidant activities of OPCs involve NF-κB and MAPK signaling pathways, thus inhibiting expression of pro-inflammatory factors and oxidation indicators. These findings provide novel experimental evidence for the further practical application of OPCs in prevention and treatment of bovine mastitis.

• Asian-Australasian Journal of Animal Sciences
• /
• 제33권1호
• /
• pp.111-119
• /
• 2020

Objective: This study was conducted to examine the effects of a mixture of pinecone oil, garlic, and brown seaweed extracts (PGBE) on milk production traits as well as physiological and ethological parameters in Holstein cows during the summer season (24 May to 03 July 2015, Korea). Methods: Among the extract combinations tested, we found that the level of 2,2'-azino-bis (3-ethylberzothiazoline-6-sulphonic acid) cation radical scavenging activity of the 0.16% PBGE complex at ratio of 1:1:1 (vol/vol) was comparable to that of the control (ascorbic acid; 1 mg/mL). Additionally, the PBGE complex reduced lipopolysaccharide-induced COX-2 expression in bovine mammary epithelial cells. Based on these findings, 40 lactating Holstein cows were used to measure the effects of PBGE complex at ratio of 1:1:1 (vol/vol) on milk production, immune response, metabolites, and behavior patterns by dividing the cows into two groups fed diets containing PGBE complex (n = 20; 0.016%/kg feed dry matter basis) or not containing PGBE complex (control, n = 20) for 40 d. Results: Results showed that PGBE complex did not influence milk composition, eating and ear surface temperature patterns, immune response, or metabolic parameters but promoted average milk yield throughout the experimental period. Additionally, a tendency of higher total antioxidant capacity and glutathione in the PGBE group was observed compared to the those in the control. When the temperature-humidity index (THI) exceeded 72 (average THI = 73.8), PGBE complex-fed cows experiencing heat stress showed increased milk yield and a tendency of increased rumination compared to the control. Conclusion: We suggest that incorporation of a combined mixture of 0.016% PGBE (1:1:1 ratio, vol/vol) to diet has the potential to improve milk yield and health status of cows under mild to moderate heat stress, denoting that it might be useful as an alternative anti-stressor in the diet of dairy cows under hot conditions.