34 PRODUCTION OF TRANSGENIC CLONED BUFFALO EMBRYOS CONTAINING OVEREXPRESSED STEAROYL Co-A DESATURASE GENE FOLLOWING EFFICIENT TRANSFECTION

Abstract

Stearoyl-CoA desaturase (SCD) is a rate-limiting enzyme that catalyses the synthesis of monounsaturated fatty acids and polyunsaturated fatty acids from saturated fatty acids, which are components of triglycerides, wax esters, cholesteryl esters, and membrane phospholipids. Alterations in phospholipid composition have been implicated in a variety of diseases including obesity and the associated metabolic syndrome. SCD also magnifies the conjugated linoleic acid (CLA) content in milk; CLA is a natural fat element, having reputed therapeutic health values including anti-carcinogenic properties. In light of this fact, this study was designed to amplify the levels of SCD at the gene level. In order to achieve the enhanced expression of SCD gene, combination of techniques were used. The somatic cells (fetal fibroblast) culture were established from ear pinnae obtained from bovine fetus procured from the abattoir and were cultured in basal medium, comprising DMEM with 10% fetal bovine serum, 1X NEAA and 1X PS antibiotics. These isolated cultured cells were transfected with a gene construct carrying buSCD gene (pAcGFPN1-buSCD) as BLGP-buSCD-BLG3′UTR-CMVP-EGFP-SV40. The buffalo fetal fibroblast cells were transfected using 3 methods: Nucleofection, Fugene and Lipofection. The successful transfection, as confirmed by PCR and Southern hybridisation, proved Nucleofection to be more efficient in transfecting cells among the techniques used, which were further maintained and selected by Geneticin (G418). These selected transfected cells were then used for nuclear transfer. Somatic cell nuclear transfer (SCNT) has provided an efficient pathway for the production of transgenic animals. Buffalo cumulus–oocyte complexes (COCs) were collected from ovaries collected from abattoir and matured in TCM-199 supplemented with 10% FBS, 5 µg mL⁻¹ FSH, and 1 µg mL⁻¹ β-oestradiol for 21 h in CO₂ incubator at 5% CO₂ in air and 38.5°C temperature with >95% relative humidity. After 21 h, these COCs were denuded and subjected to zona removal. These zona-free oocytes were manually enucleated using microsurgical blades and 2 enucleated oocytes were fused with a transgenic cell using electro cell manipulator. Further, these reconstructed embryos were activated using calcium ionophore and cultured in IVC media thereafter for 8 days. The developmental competence rate as recorded on Day 8 was 53.26 ± 1.73%, 69.87 ± 6.24%, 62.99 ± 7.15%, 42.71 ± 5.05% and 28.00 ± 3.33% for 2-cell, 4-cell, 8–16 cell, morula, and blastocyst, respectively. When observed under fluorescence microscope, the embryos showed successful expression of GFP, which can be further used for animal production or further research analysis. In conclusion, amplified SCD at gene level will result in a boost to the dairy sector as well ameliorating human health due to its crucial role in anti-cancer, anti-diabetic, reduced cardio-vascular disease, and improved immune responses.