209 PRODUCTION OF BUFFALO (BUBALUS BUBALIS) EMBRYOS CONTAINING HUMAN LYSOZYME GENE

Abstract

Lysozyme is a ubiquitous enzyme found in all major taxa of living organisms with a diverse role in human and animal health. It plays an important role as a nonspecific immune factor and anti-inflammatory factor and is a part of the innate immune system. Research on the expression of recombinant human lysozyme is, thus, potentially valuable to the dairy industry. Therefore, the present study was carried out to observe the developmental competence and quality of cloned embryos containing the human lysozyme gene. Primary fetal fibroblast cells were obtained from a slaughterhouse-derived fetus. The initial passage cells were transfected with pAchLYZ vector containing human lysozyme gene and green fluorescence protein (GFP) via nucleofection, lipofectamine, and FuGene (Roche Diagnostics, Indianapolis, IN, USA). Transfected cells were selected by adding G418 (400–800 µg µL⁻¹), which selectively killed the nontransfected cells in culture. The presence of hLYZ gene in transfected cells was confirmed by PCR amplification of this gene. For cloned embryo production, reconstructs were formed with 2 enucleated demi oocyte fused with 1 donor cell of hLYZ transfected cells or nontransfected fibroblast cells. Gene expression in the resulting embryos was assessed for apoptosis (BID, BAX, and BCL-XL) and development- (G6PD, IGF1R, and FGF) and pluripotency-related (OCT4, SOX2, and NANOG) genes. The transfection efficiency of the cells by nucleofection methods was the highest and toxicity to the cells was minimum as compared with chemical methods. Transfected cells expressed GFP within 48 to 72 h of transfection. When these cells were passaged the intensity of GFP expression was reduced and the reduced level was maintained in subsequent passages. A total of 114 and 60 reconstructed embryos were produced using transfected and nontransfected cells, respectively. When transfected cells were used, the cleavage, 4 cell, 8 to 16 cell, morula, and blastocyst rate was 62.01 ± 4.78, 43.54 ± 3.93, 27.69 ± 4.44, 24.55 ± 5.12, and 22.87 ± 5.39%, respectively, whereas rates were 73.34 ± 2.36, 68.34 ± 4.82, 59.89 ± 2.39, 46.44 ± 2.64, and 36.36 ± 2.53% for nontransfected cells, respectively. The cleavage rate had no significant (P < 0.05, Student’s t-test) difference in both type of donor cells but all other stages [i.e. 4 cell, 8 to 16 cell, morula, and blastocyst rate was significantly (P < 0.05) lower with transfected cells]. Most of the transgenic cloned embryos expressed GFP and integrated with hLYZ gene. We found that proapoptotic gene (BID and BAX) expression did not differ, whereas BCL-XL expression was significantly low (P < 0.05) in transgenic embryos. The development- (G6PD, IGF1R, and FGF) and pluripotency-related (OCT4, SOX2, and NANOG) gene expression was significantly (P < 0.05) lower in transgenic embryos. In conclusion, transgenic cloned embryo successfully developed up to the blastocyst stage in the preliminary study for producing genetically modified animal with human milk components having antimicrobial activity, which would be potentially valuable for human as well as animal health.