Double lipofection improves the reprogramming efficiency of buffalo fibroblast cells

Abstract

To date, reprogramming of ruminant cells has been a very challenging task as an efficient protocol for the production of buffalo induced pluripotent stem cells (iPSCs) has not been fully established yet. This study aims to find the most efficient method for the delivery of exogenous factors in buffalo fetal fibroblast cells to achieve maximum reprogramming efficiency. The plasmid vector pCAGMKOSiE containing mouse Oct4, Sox2, Klf4, and C-Myc was used for reprogramming buffalo fetal fibroblast cells. Nucleofection, lipofection and double lipofection were used to transfect the cells. The transfection efficiency was determined by GFP expression. The viability of post-transfected cells was determined by MTT assay. The transfected cells were cultured in stem cell media for 15 days and cells from all the groups were subjected to quantitative analysis of key pluripotency (OCT4, SOX2, NANOG, KLF4 and c-MYC) related genes. The highest transfection rate (60-65% GFP expression) was found in double lipofected cells with 0.61±0.17 viability index. The viability of cells was affected maximally by nucleofection. qPCR analysis revealed a significant elevation (p