An Optimized Mouse Embryonic Stem Cell Based Reverse Poly-Transfection Technique for Rapid Exploration of Nucleic Acid Ratios

Summary

The present protocol describes a method for reverse poly-transfection of mouse embryonic stem cells during culture with 2i and LIF media. This method yields higher viability and efficiency than traditional forward transfection protocols, while also enabling one-pot optimization of plasmid ratios.

Abstract

Due to its relative simplicity and ease of use, transient transfection of mammalian cell lines with nucleic acids has become a mainstay in biomedical research. While most widely used cell lines have robust protocols for transfection in adherent two-dimensional culture, these protocols often do not translate well to less-studied lines or those with atypical, hard-to-transfect morphologies. Using mouse pluripotent stem cells grown in 2i/LIF media, a widely used culture model for regenerative medicine, this method outlines an optimized, rapid reverse transfection protocol capable of achieving higher transfection efficiency. Leveraging this protocol, a three-plasmid poly-transfection is performed, taking advantage of the higher-than-normal efficiency in plasmid delivery to study an expanded range of plasmid stoichiometry. This reverse poly-transfection protocol allows for a one-pot experimental method, enabling users to optimize plasmid ratios in a single well, rather than across several co-transfections. By facilitating the rapid exploration of the effect of DNA stoichiometry on the overall function of delivered genetic circuits, this protocol minimizes the time and cost of embryonic stem cell transfection.

Introduction

Delivery of DNA and RNA into mammalian cells serves as a core pillar of biomedical research¹. A common method for introducing exogenous nucleic acids (NA) into mammalian cells is through transient transfection^2,3. This technique relies on mixing NA with commercially available transfection reagents capable of delivering them into the recipient cells. Typically, NA is delivered via forward transfection, where cells adhering to a two-dimensional surface receive the transfection complex. While forward transfection for the most common established cell lines is robust and protocols are well-published, more niche cell types with non-monolayer morphologies do not transfect easily, limiting the amount of NA that can be delivered and the number of cells that receive it.

Pluripotent stem cells (PSCs) serve as an attractive model for understanding development and as a tool for regenerative medicine, given their ability to divide indefinitely and produce any bodily cell type. For mouse PSCs (mPSCs), routine in vitro culture conditions with 2 inhibitors and LIF (2i/LIF) maintain a dome-like colony morphology, directly limiting the number of cells exposed to a forward transfection^4,5,6. To address this, a reverse transfection can be performed: cells are added to a dish containing media and transfection reagent, rather than adding transfection reagent to adherent cells⁷. While this increases the number of cells exposed to the reagent, it also requires the cells to be passaged and transfected concurrently.

Moving beyond simple single-NA transfections, researchers often aim to deliver several NA constructs into a population of cells in vitro. This is typically achieved through a co-transfection, where the NAs are mixed at a given ratio (1:1, 9:1, etc.) and are then combined with the chosen transfection reagent⁸. This yields a mix of NAs and reagent that preserves the original ratio of NAs to one another – while cells in the treatment may receive different amounts of this mix, they all receive the same ratio⁹. While this is advantageous when the desired ratio of parts is known, determining this ratio ahead of time can be labor-intensive, with each ratio constituting a different condition. One alternative is to perform a "poly-transfection," where individual NAs are mixed with the transfection reagent independently from one another⁹. By combining transfection complexes containing individual NAs (rather than combining NAs before creating the complexes), researchers can explore a wide array of NA stoichiometries in a single transfection experiment⁹. This is particularly valuable in cases where the products of several NAs are expected to interact with one another, such as with inducible transcription systems or systems with feedback built in^1,10,11. However, to do so effectively, a high transfection efficiency is needed. Indeed, as the number of unique transfected NAs increases, the probability of a given cell receiving all of the desired NAs decreases exponentially^{9, 12}.

The following report describes a reverse transfection protocol for mPSCs using a cationic lipid-based transfection reagent, in which cells are exposed to the reagent-NA mix for a maximum of 5 min to maximize viability and minimize the time outside of typical culture conditions. Comparing this protocol to the standard forward transfection of these cells demonstrates a higher transfection efficiency and an increase in the total number of surviving transfected cells. By combining this reverse transfection with a three-plasmid poly-transfection involving simple fluorescent reporters, an expanded potential to screen NA ratios with high transfection efficiency is demonstrated.

Protocol

1. Preparation of reagents for mPSC culture Prepare N2 supplement. In non-sterile conditions, prepare the following stock solutions (step 1.1.1.1) in a chemical safety fume hood. Add the solid powder of each chemical to a pre-weighed 50 mL conical tube. Weigh each tube after adding the powder and add an appropriate amount of solvent to achieve the concentrations below. For one batch of media, prepare the minimum amount listed. NOTE: The following reagents are hazardous and…

Representative Results

Both forward and reverse transfections rely on the interaction between the cell membrane and incoming transfection reagent-DNA complexes, allowing the delivery of NA to the recipient cells. Where these techniques differ is the state of the cell upon delivery – while DNA is typically delivered to adherent cell monolayers in traditional forward transfection, reverse transfection instead relies on having the reagent-DNA complex meet the cells while in a single-cell suspension. This difference can be particularly crucial in …

Discussion

A key reason for the widespread adoption of transfection protocols is their reproducibility and accessibility; however, these protocols do require optimization across experimental contexts. Not mentioned above is the standard testing required when attempting to transfect a new cell line for the first time. First, the choice of transfection reagent is key, as commercially available reagents are not one-size-fits-all and will vary in the efficiency of NA delivery viability across cell types. Additionally, finding the ideal…

Materials

Accutase	MilliporeSigma	SCR005
Apotransferrin	MilliporeSigma	T1147-500MG
B27 supplement	ThermoFisher Scientific	17504044
Beta-mercaptoethanol	ThermoFisher Scientific	21985023
BSA fraction V (7.5%)	Gibco	15260-037
CHIR99021	MilliporeSigma	SML1046-25MG
DMEM-F12	MilliporeSigma	D6421-24X500ML
Flow cytometry standardization beads	Spherotech	URCP-38-2K
Gelatin	MilliporeSigma	G1890
GlutaMAX supplement	ThermoFisher Scientific	35050061
Insulin	Gibco	12585-0014
Lipofectamine 2000	Invitrogen	11668-019	Transfection reagent
Neurobasal media	ThermoFisher Scientific	21103049
OptiMEM	Invitrogen	31985-070
PD0325901	MilliporeSigma	PZ0162-25MG
Progesterone	MilliporeSigma	P8783	Chemical hazard – consult local safety guidelines, ensure proper PPE is worn, and work with the solid powder form only in a chemical fume hood
Putrescine	MilliporeSigma	P6780	Chemical hazard – consult local safety guidelines, ensure proper PPE is worn, and work with the solid powder form only in a chemical fume hood
Recombinant mLIF	BioTechne	8878-LF-500/CF
Sodium selenite	MilliporeSigma	S5261-25G	Chemical hazard – consult local safety guidelines, ensure proper PPE is worn, and work with the solid powder form only in a chemical fume hood
Trypsin-EDTA	ThermoFisher Scientific	25200056