Vol. 10, Issue 6 (2021)

Author(s):
Richa Arora, Arpit Tyagi, Waseem Akram Malla, Shikha Saxena, Sonalika Mahajan, Basavaraj Sajjanar and Ashok Kumar Tiwari

Abstract:
Various gene expression studies based on transfection of DNA in eukaryotic cells requires suitable transfection method. After transfection procedure selection, there is need to optimize transfection efficiency which varies in each cell type depending on transfection conditions. There are few significant transfection conditions like number of cells seeded 24 hrs before transfection, cell confluence at the time of transfection, optimal transfection reagent/DNA concentration ratio, amount of media added etc., which are required to be optimized individually in each cell type to attain highest transfection efficiency. Transfection with chemical method by using cationic lipids like lipofectamine is commonly used transfection method. Reporter gene like GFP cloned in any vector can be used to optimize transfection for attaining maximum transfection efficiency. In our present study we optimized Lipofectamine 3000®/pVIVO1.GFP plasmid concentration ratio to attain highest transfection efficiency in 4T1 mice mammary tumor cells. Non structural gene of Canine Parvovirus is an oncolytic viral gene reported to be cytotoxic selectively to cancer cells without harming healthy cells. NS1 gene of CPV2 cloned in pVIVO1 vector is already available in our laboratory. To identify different oncolytic mechanisms of CPV2.NS1, there was need to transfect this gene in 4T1 cells and therefore we used GFP as reporter gene cloned in same vector of CPV2.NS1 (pVIVO1.GFP) so as to optimize for Lipofectamine/DNA ratio depicting highest transfection efficiency. The concentration of plasmid DNA (pVIVO1.GFP) was optimized to 3µg in 6 well plate with 6µl of Lipofectamine 3000® by our study. In this concentration of plasmid DNA, the 4T1 cells were showing highest transfection efficiency.