Comparing transfection methods for enhanced gene delivery: A study of Calcium Chloride, Polyethylenimine, and JetPrime in HEK 293T Cells using mEGFP and mCHERRY reporters

Hatice Esenkaya

doi:10.26900/hsq.2717

Authors

Hatice Esenkaya Karolinska Institutet / Sweden 0000-0001-9357-4975

10.26900/hsq.2717

Abstract

Transfection is a fundamental technique for introducing foreign nucleic acids into eukaryotic cells. Plasmids are commonly transfected to deliver genes of interest, often alongside fluorescent reporter tags like modified green fluorescent protein (mEGFP) and mCHERRY to enable real-time tracking of gene expression and protein localisation. These elementary studies facilitate studies of gene functions, protein expression, and cellular processes which are critical to advancing biochemical engineering strategies including genetic modifications, recombinant protein production and gene therapies. Despite this, there is no standardised optimal protocol for eukaryotic transfection. Here we compare the efficiency and cytotoxicity of three transfection methods: Calcium chloride (CaCl₂), Polyethylenimine (PEI), and JetPrime using HEK 293T cells and plasmids encoding mEGFP and mCHERRY. Transfection efficiency was evaluated through fluorescence imaging at 24 and 48 hours. JetPrime showed the highest efficiency after 24 hours, although its expression was cytotoxic at 48 hours. PEI achieved consistently high transfection rates with minimal cytotoxicity across both time points, making it the most reliable method. CaCl₂ yielded moderate expression levels but was least efficient of the three, particularly at 24 hours. These insights underscore the importance of optimizing transfection methods to enhance cellular outcomes and can enhancing the precision and efficacy of future biotechnological applications.

Keywords:

Transfection plasmids fluorescent reporter tags cellular imaging gene engineering

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References

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