Team:REC-CHENNAI/Project-Demonstration
PROJECT DEMONSTRATION
The first step towards achieving efficient transfection involves the optimization of the N/P ratio of the nucleotide cargo and delivering peptide. The N/P ratio is determined by the moles of amine groups in the peptide and the moles of phosphate groups in the DNA. The experiment involving optimization of the N/P ratio for improved transfection efficiency can be found here.
Click here for Improved Transfection Efficiency N/P ratioWe observed that the N/P ratio of 0.5 gave the highest intensity of fluorescence, which can be attributed to the higher extent of complexation resulting in efficient transfection. Hence, this optimized N/P ratio was fixed for further validation experiments.
Scramble (negative control), shRNA (positive control), and shRNA-like siRNA (our biobrick) were individually mixed with CPP at the optimum N/P ratio of 0.5 as concluded from experiment 1. These nucleotides were also mixed with Lipofectamine 2000 in the ratio of 1:2 to form liposome complexes. Both the sets of mixtures were used to transfect HeLa cells. The efficiency of cargo delivery was determined by quantifying the fluorescence intensity in Arbitrary Fluorescence Units (AFU). It can be observed that no fluorescence signal is emitted from cells transfected with scramble or shRNA owing to the fact that these two constructs have no fluorophore. Cells treated with only siRNA also showed no fluorescence signal despite bearing fluorophore, accounted by the reason that there’s no delivery agent in this experimental set-up to introduce these nucleotides into the cell. Between the cells transfected with siRNA using Lipofectamine 2000 and the CPP BP100-(KH)9, the ones transfected with the CPP reflected significantly higher fluorescence than the ones transfected by Lipofectamine 2000, thereby confirming that the CPP BP100-(KH)9 has increased delivery efficiency when compared to Lipofectamine 2000.
Real-Time Quantitative Reverse Transcription PCR (qRT-PCR) was done to evaluate and thus compare the efficient silencing of the gene expression by Lipofectamine 2000 and the chimeric CPP BP100-(KH)9
Finally, qRT-PCR was done to compare the relative gene expression of Survivin using GAPDH as control. It was found the siRNA delivered by CPP efficiently silenced gene expression at mRNA level, when compared to that delivered by Lipofectamine 2000, which confirms that our part works efficiently when compared to Lipofectamine 2000. Furthermore, comparable levels of gene silencing was observed between our shRNA-like siRNA and the shRNA construct we synthesized by referring the literature. (Wang et al., 2017)
These results, in conjunction with our part characterization results, together validate the fact that our system works efficiently, and provides a non-toxic and cost-effective replacement for Lipofectamine 2000.
Wang YL, Shao X, Wang F, et al. Intron-specific shRNA-mediated downregulation of survivin and promotion of apoptosis in HeLa cells. Oncol Lett. 2017;14(5):5927–5933. doi:10.3892/ol.2017.6996
