Journal of
Developmental Biology and Tissue Engineering

  • Abbreviation: J. Dev. Biol. Tissue Eng.
  • Language: English
  • ISSN: 2141-2251
  • DOI: 10.5897/JDBTE
  • Start Year: 2009
  • Published Articles: 42

Full Length Research Paper

Nucleofection an efficient non-viral transfection technique forIFN-t-EGFP gene expression study in Sahiwal cattle fibroblast cells

Anand Laxmi N.1, Gunjan G.1 and Prateesh M.2*
1DCP, NDRI, Karnal, India. 2IVRI, Barrielly, India.
Email: [email protected]

  • Article Number - 8CE855010393
  • Vol.3(3), pp. 23-32, March 2011
  •  Accepted: 20 December 2010
  •  Published: 31 March 2011

Abstract

Viral based techniques were considered to be the most efficient systems to deliver DNA into fibroblast cells as they show high transgene expression in many cellular models. Viral approaches are complicated by immune response, intracellular trafficking potential mutations and genetic alterations due to integration. The nucleofector TM technology is electroporation based gene transfer technique which has been proved to be an efficient tool for transfecting primary cells and hard to transfect cell lines. The present study was designed to examine Sahiwal fibroblast cell to act as competent donor cell in gene expression studies. Using a green fluorescent protein reporter vector, a high transgene expression level was obtained using U-12 and U-23 pulsing programs: 45 and 70% respectively. Cell recoveries and viabilities were 90.5 and 95% respectively for U3-23 program. Overall transfection efficiency was 60% as observed on evaluation by flowcytometry. Further, the cells confirmed to be positive for gene expression when subjected to PCR, RT- PCR and, flow cytometry analysis using pGFP repoter other than supplied by Amaxa system. Hence, the cells were transfected with bIFN-t---GFP reporter gene construct by nucleofection technique and similarly, cells were evaluated for expression of gene by fluorescence microscopy. A sixty percent of cells were positive for fuorescence on enumeration of transfected cells by fluorescence microscopy 72 h post transfection. Further it was confirmed by PCR technique using primers for IFN-t gene. The cell cultures when analysed for IFN-t protein expression, the protein could be detected by silver staining of SDS-PAGE gels. Reports are available where this technology has been successfully applied for the transfection of other cells like monocytic cell lines and neural stem cells. This technique can be further utilised for transfection of genes of economic importance in primary cell lines which be in turn can be utilised for production of transgenic embryos. Through out the study the transfected cell cultures were negative for apoptosis. The Sahiwal fibroblast cells culture system can be utilized for transfection and gene expression studies. Green Fluorescent Protein acts as a useful visual indication system for gene insertion or targeting studies, when conjugated to the gene of interest

 

Key words: Transfection, gene, fibroblast cell.

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