Incorporating the gene of interest has always been keen interest to humans. Many potential application arise from transgenic animals, right from harvesting desired protein to creating species with enhanced ability. One such important enzyme Stearoyl Co-A Desaturase (SCD), which is involved in catalysis of monounsaturated fatty acids and polyunsaturated fatty acids from saturated fatty acids. It has also been implicated in diseases including obesity and the associated metabolic syndrome. Thus overexpressing buSCD in bovine species can really ameliorate human health, but the method of transfection has always been the limiting factor. In the present study, we have tried to study three non-viral methods: Nucleofection, Lipofection via Lipofectamine 3000 and Fugene HD. Initially, the somatic cell culture was established by culturing fetal ear skin (fetal fibroblast) explant in basal medium, comprising DMEM with 10% fetal bovine serum. To these somatic cells, our constructed vector, i.e. pAcGFPN1-buSCD was transfected using the above mentioned methods and non-transfected cell of same passage were used as control in the experiment. The efficiency was measured after 48-72 h of transfection via Fluorescence microscopy, Dye exclusion method (Trypan Blue-Live dead assay) and Real-time analysis. Each experiment was repeated 3 times and statistically analysed in Graphpad Prism. GFP expression in transfected Cells showed 62.4 %± 2.3in nucleofection, 48.8 % ±1.48 in Lipofectamine 3000 and50.7 % ±2.48 in Fugene HD. Live-Dead assay revealed, cells transfected by nucleofection (76.42 ± 1.53) were significantly more alive (P<0.05) in comparison to Lipofectamine 3000 (67.34 ± 4.04)and Fugene HD(63.67 ± 1.53) respectively. Similar results were also shown by real-time analysis, in which apoptosis markers (CASPASE3 and p53) were significantly less expressive in nucleofected cells in comparison to cells transfected by Lipofectamine 3000 and Fugene HD. Overall, it can be inferred that nucleofection is better method of transfection as it shows lesser mortality and higher level of transfection in transfecting pAcGFPN1-buSCD, which can be further used for creating transgenic animals.
Bibliographical noteFunding Information:
This work was supported by Department of Biotechnology, Government of India, New Delhi, India.
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- Buffalo fetal fibroblast
- Fugene HD
- Lipofectamine 3000