Increasing the efficiency of transfecting vectors encoding KZFP genes into mouse embryonic stem cells

Document Type

Article

Publication Date

2025

Keywords

JMG

Abstract

Future downstream studies to understand the regulatory genes responsible for cleft lip palate (CLP) in the A/WySn mouse model require a large supply of mouse embryonic stem cells (mESCs) transfected with large candidate regulatory KRAB zinc finger protein (KZFP) genes. However, current mESC transfection protocols yield on average 2-5%. This originates from their cohesive colonial nature, the 2i standard media they culture in, the conventional method of transfecting cells through adhesive cultures, and the large DNA cargo being transfected. Previous chemical transfection protocols could only yield 2-5% transfected mESCs, and yet we require a greater yield for large scale research into Clf1 and Clf2 roles in CLP development. Our project will experiment on factors that could impact yield rates, such as liposomal and non-liposomal transfection reagents, vector sizes, reagents supplemented through adhesive and/or suspensions, and modifications to the 2i standard ESC culture media. We concluded an optimal liposomal- based transfection protocol utilizing Lipofectamine 2000 in a trypsinized non-antibiotic cellular suspension under a 5-minute incubation sequence offers an average of 10-30% transfection yield. This improved transfection efficiency method will prove vital for future downstream studies of early embryonic development of CLP in A/WySn mouse strains.

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