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

Kieran Maheden; Karen Hwang; Ipek Egilmez; Nika Shakiba

doi:10.3791/65766

JoVE Journal > Developmental Biology

Developmental Biology

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

Published: December 08, 2023

doi:

10.3791/65766

Kieran Maheden¹, Karen Hwang¹, Ipek Egilmez¹, Nika Shakiba¹

¹School of Biomedical Engineering,University of British Columbia

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 transfection²^,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 transfection⁴^,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 in¹^,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 exponentially⁹^, 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.
1. 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 should be handled in accordance with local chemical safety guidelines. Ensure proper PPE when handling, and only work with the solid powder forms of these chemicals in a chemical fume hood to prevent inhalation.
  1. Minimum of 0.05 mL of sodium selenite in water at 0.518 mg/mL, 0.5 mL of putrescine in water at 160 mg/mL, 0.165 mL of progesterone in 100% ethanol at 0.6 mg/mL (see Table of Materials).
  2. Store solutions at -20 °C for up to 2 years.
2. In a biosafety cabinet (BSC), add the following to 58.035 mL of DMEM-F12.
  1. 0.05 mL of a 0.518 mg/mL sodium selenite solution, 0.5 mL of a 160 mg/mL putrescine solution, 0.165 mL of a 0.6 mg/mL progesterone solution.
  2. Filter sterilize the above mixture with a 0.22 µm filter.
3. Still in the BSC, prepare a 100 mg/mL solution of apotransferrin.
  1. Add 5 mL of DMEM-F12 to 500 mg of apotransferrin (see Table of Materials). Slowly resuspend and wash the container, avoiding bubbles.
  2. After resuspending and removing as much as possible with 5 mL, add it to the selenite/putrescine/progesterone solution. Take more of the previous solution and wash the apotransferrin bottle, getting as much of it out as possible.
4. Add 5 mL of 7.5% BSA fraction V (see Table of Materials) to the solution.
5. Mix and aliquot 6.875 mL per tube. Store aliquots at -80 °C for up to 1 year.
6. When using the above N2 aliquots to make N2B27 media, thaw the desired aliquot and add 3.125 mL of a 4 mg/mL insulin solution (see Table of Materials) to the 6.875 N2 for 10 mL of 100x N2.
Prepare N2B27 basal media.
1. Thaw N2 and B27 supplements in a 4 °C fridge for a few hours or overnight.
2. In a BSC, mix 484.5 mL of DMEM-F12 and 484.5 mL of Neurobasal media (see Table of Materials) in a sterile 1 L bottle.
3. Add 10 mL of B27 and 10 mL of N2 supplement to the media.
4. Add 1 mL of 55 mM beta-mercaptoethanol (see Table of Materials). Add 10 mL of 100x glutamax. Mix vigorously by swirling the bottle.
5. Aliquot the desired volume per aliquot (typically 100 mL) and store at -80 °C for up to 1 year. Store in use after thawing at 4 °C for up to 3 weeks.
Prepare NBiL media.
1. Thaw a 100 mL aliquot of N2B27 basal media from -80 °C in the 4 °C fridge.
2. In a BSC, add LIF (see Table of Materials) to a final concentration of 10 µg/L.
3. Add CHIR99021 (3 µM final) and PD0325901 (1 µM final) (see Table of Materials). Mix well with a 50 mL serological pipette. Store at 4 °C for up to 2 weeks.
Prepare 0.2% gelatin solution.
1. Transfer 500 mL of double-distilled H₂O into a 1 L glass bottle.
2. Measure out 1 g of gelatin (see Table of Materials) and add it to the water. Mix by shaking the bottle until mostly dissolved.
3. Autoclave the gelatin and water mixture at 15 psi, 121 °C for 35 min. Once cooled, filter sterilize it with a 0.22 µm filter in a BSC. Store at 4 °C.
Prepare wash media.
1. In a BSC, mix 160 µL of 7.5% BSA per 10 mL of DMEM-F12.
2. Scale the above and prepare in large quantities for ease of future use (e.g. 8 mL of 7.5% BSA to 500 mL of DMEM-F12). Store at 4 °C.

2. Preparation of reagents for mPSC poly-transfection

NOTE: The following values are provided for a single well of a 24-well plate. Values can be scaled accordingly. The sequences of all the DNA/plasmids are detailed in Supplementary File 1.

Calculate the volume of DNA solution needed per treatment.
1. Prepare 500 ng of DNA per well/condition.
  1. If transfecting a single plasmid with a DNA solution of 100 ng/µL, prepare one tube with 5 µL of DNA.
  2. If transfecting two plasmids at 100 ng/µL each, prepare two tubes with 2.5 µL in each, one for each plasmid.
In a BSC, perform the following: For each 500 ng transfection treatment (see Table of Materials), aliquot 25 µL of OptiMEM into a 1.5 mL tube.
1. Scale this accordingly – for the example above, prepare two tubes, each with 250 ng of DNA (2.5 µL) and 12.5 µL of OptiMEM.
Add the required volume of DNA for that treatment to the OptiMEM in the tube.
For each tube with 500 ng DNA and OptiMEM, create a matching tube with another 25 µL of OptiMEM and 1 µL of cationic lipid-based transfection reagent.
1. Again, these values must be scaled accordingly. For the above example, prepare two tubes, each with 12.5 µL of OptiMEM and 0.5 µL of transfection reagent.
  NOTE: When scaling values, use the mass of DNA added, not the volume required for that amount of DNA. Reactions with transfection reagent and DNA can be scaled (e.g., if 5 wells are to be transfected with the same DNA). Keep the ratios of reagents the same, but scale accordingly (e.g., 1000 ng DNA: 50 µL OptiMEM: 2 µL transfection reagent: 50 µL OptiMEM).

3. Preparation of mPSCs for transfection

NOTE: For reverse transfection, prepare the culture vessel and passage the mPSCs directly before adding the transfection reagents. For forward transfection, passage and plate the cells 12-18 h prior to transfection to allow the cells to adhere to the plate.

Prepare a 0.2% gelatin-coated cell culture vessel.
1. Plan the number of wells needed for the transfection (one well of a 24-well plate per treatment/group).
2. Add 200 µL of the 0.2% gelatin solution to each of the desired wells in the 24-well plate.
3. Gently shake the plate to evenly spread the solution, ensuring it covers the entire bottom of the well.
4. Allow the wells to coat for a minimum of 15 min at room temperature.
5. Using a vacuum aspirator, carefully remove any leftover gelatin from the wells (tilt the plate to ensure removal of all liquid without scraping off the gelatin layer).
6. Add 0.5 mL of NBiL media (step 1.3) to each well and leave the plate in a tissue culture incubator to prewarm.
Passage mPSCs.
1. Aliquot 30 mL of wash media (step 1.5) into a 50 mL conical tube.
2. Aspirate the old media from the tissue culture dish of mESCs.
  NOTE: Several aspiration techniques are permissible. Whatever technique is chosen, ensure that cells do not remain dry for extended periods (approximately 30 s maximum).
3. Add the required amount of commercially available cell detatchment medium (1.5 mL for a 10 cm dish, scale accordingly, see Table of Materials).
4. Wait for 3-5 min before pipetting up and down 15-20 times with a P1000 pipette to obtain a single-cell suspension. View cells under a microscope to ensure a single-cell suspension.
5. Transfer the cells in the detatchment medium into the 50 mL tube containing wash media.
6. Centrifuge at 300 x g for 5 min at room temperature. Aspirate wash media, ensuring no residual liquid remains in the tube.
7. Resuspend the pellet in a small volume of wash media (~300 µL). Count the cell suspension using a hemocytometer to obtain the cell density.

4. Reverse transfection of mPSCs

Prepare poly-transfection reagents according to step 2.
Prepare the cell culture vessel and passage mPSCs according to step 3.
Aliquot 200 µL of NBiL media to 1.5 mL tubes, one for each treatment.
Add 26 µL of the OptiMEM:transfection reagent mixture to the DNA:OptiMEM mixture. Mix the reagents quickly and vigorously by pipetting approximately 10 times. Allow the mixture to incubate at room temperature for 5 min.
Based on cell density, transfer the required volume of cells from the 300 µL cell suspension to the 200 µL NBiL tubes to obtain 25,000 cells per tube.
After 5 min of incubation of the Lipofectamine 2000 (L2K, transfection reagent) and DNA mixture, add it to the 1.5 mL tube of cells and mix well by pipetting. Allow the cells to incubate with the reagent for 5 min at room temperature.
Centrifuge at 300 x g for 5 min at room temperature. Pipette out the liquid, leaving approximately 50 µL.
Resuspend the cell pellet with 100 µL of NBiL media. Transfer the cells to the plate and place the plate in the incubator. Change the media 24 h later with fresh NBiL media.

5. Forward transfection of mPSCs

12-18 h prior to transfection, prepare the cell culture vessel and passage mPSCs according to step 3.
Based on cell density, transfer the required volume of cells from the 300 µL cell mixture to seed 25,000 cells per well. Place the plate in the incubator.
1 h prior to transfection, aspirate the media from all wells and replace it with 0.5 mL of NBiL media. Place the plate in the incubator.
At the time of transfection, prepare mPSC poly-transfection reagents according to step 2.
Add 26 µL of the OptiMEM:transfection reagent mixture to the DNA:OptiMEM mixture. Mix the reagents quickly and vigorously by pipetting approximately 10 times. Allow the combined mixture to incubate at room temperature for 5 min.
Add the combined mixture to the wells dropwise. Place the plate in the incubator. Change the media 24 h later.

6. Flow cytometry

Aspirate the media from the transfected 24-well plate 48 h after transfection.
Add 200 µL of trypsin to each well, swirl, and incubate for 30 s at 37 °C. Tap the sides of the plate and add 200 µL of ice-cold FACS buffer (2% FBS in PBS).
Open and label a V-bottom 96-well plate, starting from A1. Mix the contents of each well on the transfected plate to dislodge cells and make single-cell solutions with a P200 pipette. Transfer 200 µL from each well to the appropriate well on the 96-well plate.
Add 15 mL of FACS buffer to a 50 mL reagent reservoir. Centrifuge the plate at 300 x g for 5 min at room temperature. Discard the supernatant by inverting the 96-well plate into the sink with a fast and smooth movement.
Use a multichannel pipette to resuspend the pellets on the 96-well plate in 150 µL of FACS buffer from the reagent reservoir. Repeat steps 6.4-6.5 twice. Place the 96-well plate in an ice-filled box protected from light.
Run the samples through a flow cytometer to obtain the population distributions of the fluorescent reporters.
1. Ensure the cytometer is appropriate in terms of laser and filter combinations for the experiment to be performed (e.g., do not use an infrared reporter if it is not possible to excite or detect in the far-red spectrum).
2. Include additional samples for standardization beads to allow for MEFL conversion of data during analysis. Ensure that enough cells are collected per sample for appropriate statistical analysis⁹.
Convert fluorescent units from the raw cytometer files and analyze the data using any of several methods, such as Python- or MATLAB-based pipelines or commercially available software⁹^,¹³.

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 situations where cells do not grow as a uniform, flat monolayer and instead adopt a more domed or colony-like morphology, as with mPSCs. Additional variations on traditional transfection can be adopted, such as performing a poly-transfection instead of a co-transfection. This modification changes the relative distributions of each DNA species in a given treatment, allowing researchers to quickly explore the relationship between phenotype and the dosage of their plasmids. When performing these experiments, it is also critical to perform standardization and quality control of the cytometer itself. Fluorescent units for several of the experiments below have been normalized to a fluorescent bead standard to allow accurate comparison across experimental days (Supplementary Figure 1) according to established protocols¹³. Cells were gated manually as shown (Supplementary Figure 2).

Given the colony morphology of mPSCs, traditional forward transfection methods would be limited by the number of cells exposed directly to the surrounding media. When compared to reverse transfections, forward transfections had a significantly lower proportion of cells positive for the marker (>3-fold less, p < 0.05, Figure 1A) despite having a non-significantly lower amount of DNA delivered on average to the population of cells (~1.3 fold, Figure 1B).

Interestingly, incubation time significantly affected the proportion of cells that were positive for the reporter, but not the average reporter expression in the positive cells (Figure 2A,B). Incubating cells for as long as 20 min was seen to increase the percentage of cells positive for the reporter, with longer than 40 min decreasing this percentage. Critically, while performing the reverse transfection by adding the reagent directly into the well with passaged cells gave the highest %-positive and mean expression levels, it also resulted in a lower number of surviving cells at the chosen endpoint. While an increase in transfection efficiency can be seen when incubating beyond the suggested 5 min for this protocol, caution is advised as prolonged perturbation may have consequences for stem cell state and differentiation capability⁶. To clarify the impact of poly- and co-reverse and forward transfections on the overall growth of cells following transfection, we performed cell counts 48 h after transfection (Figure 3). For both poly- and co-transfection, performing a forward transfection significantly decreased the number of cells present at the chosen endpoint when compared to reverse transfections. No significant difference was observed when comparing between poly- and co-transfection within reverse or forward transfections.

When comparing forward and reverse poly-transfection, there is an observably higher transfection efficiency in the case of reverse transfections (Figure 4A). With three fluorescent reporters being delivered to cells, the number of triple-positive cells resulting from forward transfections is significantly lower than those seen in the reverse treatments. The result of the difference in transfection efficiency can also be seen in the resulting distributions for the poly-transfected populations (Figure 4B). While the total range of ratios explored by both is roughly the same, the overall number of cells across the distribution is lacking in the case of the forward poly-transfection. When comparing these conditions to the co-transfections, a difference in DNA ratios delivered can be seen: while co-transfection typically results in a delivery of approximately 1:1:1, the poly-transfection delivers the reporters across a several log-fold range.

When observing the overall distribution of DNA ratios for a reverse poly-transfection, the advantage of the increased efficiency can be appreciated (Figure 4B). While both forward and reverse transfection give a good representation of ratios that are close to the actual mixed ratio of DNA (1:1:1), the reverse condition has an expanded dynamic range of ratios that are well represented.

Figure 1: Comparison of transfection efficiency between forward and reverse transfections in mouse embryonic stem cells. Reverse transfected cells were treated with GFP vector-transfection reagent complexes for 5 min before a single wash and plating. Efficiency was quantified via flow cytometry 48 h post-transfection. 25,000 cells were treated with 1 µL of cationic lipid-based transfection reagent and 500 ng of DNA for all conditions. (A) Comparison of percent-positive for cells transfected via reverse or forward transfection. GFP-positive cells were determined by gating against non-transfected cells in addition to identifying non-transfected cells in each treatment in the mixed population. (B) Comparison of the mean fluorescence intensity of populations of cells transfected positive for GFP via forward or reverse transfection. Error bars represent mean and 1 standard deviation. Data corresponds to 3 independent replicates, each from a different passage number. Significant differences according to two-tailed t-tests are indicated (*p < 0.05). Please click here to view a larger version of this figure.

Figure 2: Titration of incubation time for reverse transfections of a GFP vector into mouse embryonic stem cells. Cells were mixed with 1 µL of cationic lipid-based transfection reagent and 500 ng of DNA for various amounts of time before being washed and plated. Overnight treatments consisted of cells being plated directly into wells containing transfection reagent-DNA complexes, with a media replacement for the treatment 18 h later. GFP expression was quantified via flow cytometry. (A) Comparison of the mean fluorescence intensity of cells transfected positive for a GFP vector. (B) Comparison of the percentage of cells in each treatment that expressed the GFP reporter following various durations of incubation with GFP vector- cationic lipid-based transfection reagent complexes. GFP-positive cells were determined by gating against non-transfected cells in addition to identifying non-transfected cells in each treatment in the mixed population. Error bars represent mean and 1 standard deviation. Data corresponds to 3 independent replicates, each from a different passage number. Significant differences according to two-tailed t-tests are indicated (*p < 0.05). Please click here to view a larger version of this figure.

Figure 3: Comparison of abundance between co- and poly- reverse and forward transfections. Cells were transfected with 500 ng of vector DNA and 1 µL of cationic lipid-based transfection reagent then harvested for hemocytometer live cell counts 48 h later. Cell counts were normalized to non-transfected controls. Error bars represent mean and 1 standard deviation. Data corresponds to 3 independent replicates, each from a different passage number. Significant differences according to two-tailed t-tests are indicated (*p < 0.05). Please click here to view a larger version of this figure.

Figure 4: Three reporter transfection of mouse embryonic stem cells comparing forward and reverse poly- and co-transfection techniques. Cells were treated for 5 min at room temperature with 1 µL of cationic lipid-based transfection reagent and 500 ng of DNA (167 ng each of GFP, RFP, and BFP expression vectors). 48 h later cells were analyzed via flow cytometry. (A) Proportion of all cells analyzed that were positive for all three reporters. Reporter-positive cells were determined by gating against non-transfected cells in addition to identifying non-transfected cells in each treatment in the mixed population. Error bars represent mean and 1 standard deviation. Data corresponds to 3 independent replicates, each from a different passage number. Significant differences according to two-tailed t-tests are indicated (*p < 0.05). (B) Distribution of reporter expression within the triple-positive population. BFP and RFP signals were normalized to GFP signals for each given cell analyzed, then plotted per condition to visualize the ratios of DNA vector per cell. Plot distritubtion was coloured as a pseudocolour plot, dependent on the density of cells at a given space. Please click here to view a larger version of this figure.

Supplementary Figure 1: Calibration curve. Standard calibration curve used to normalize arbitrary fluorescent units in the GFP channel (B525_FITC_A) to molecules of equivalent fluorescein (MEFL). Standard curves and normalization were generated and performed using a flow cytometry data analyzer. Please click here to download this File.

Supplementary Figure 2: Example gating strategy for determining the percent-positive for a single marker. Cell plots are first gated for cells via (FSC-A vs. SSC-A) and then cells are gated for doublets (FSC-W vs. FSC-H). Percent positive gates are generated by using a non-transfected population and gating such that 1% of this population is within the gate. Please click here to download this File.

Supplementary File 1: Sequences of the DNA/plasmids used in the present study. Please click here to download this File.

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 amount of NA and transfection reagent, as well as their optimal ratio, requires testing. Often following the transfection reagent supplier-recommended optimization steps is sufficient to arrive at the ideal amount of both NA and transfection reagent.

When a given transfection has failed, the first consideration should be the suitability of the reagents used: consider whether the NA is validated and sequenced, whether the cells are in an optimally viable state before the transfection, whether the transfection reagent has been lot-tested or compared to other more cell-specific reagents. Often times a critical control is the inclusion of a vector expressing GFP under the control of a tested and validated promoter, as promoters are often known to be cell-specific, with different strengths in different cell lines¹⁴. Outside of user technical error, once a given transfection protocol and NA has been optimized, large deviations in experimental outcomes can often be traced to issues with a given reagent.

While the use of poly-transfection enables several types of single-pot experiments that are unfeasible with traditional co-transfections, it is not always the clear best choice. In cases where the ratio of several NA must be titrated, or where the presence of one NA will influence the others in a dose-dependent manner, poly-transfection makes it possible to conduct rapid design-build-test cycles with NAs⁸^,⁹. On the other hand, poly-transfections are not well-suited to applications where single-cell analysis, such as flow cytometry, is not being performed. In these cases, a co-transfection approach to produce a homogenous population may be more desirable. Additionally, for cells that are hard to transfect regardless of optimization, poly-transfections may not yield a suitable number of cells across the entire distribution for downstream analysis without heavily scaling up the experiment.

Finally, some cell lines do not tolerate transfection, regardless of the reagent or optimization. Unfortunately, reports on such lines are typically not published, limiting information on the transfectability of an individual line. In such cases, other NA delivery methods may be required, such as electroporation or virus-mediated delivery. When possible, however, expanded transfection protocols such as reverse poly-transfection techniques as described above open up a new regime for researchers, building on time-tested reagents and protocols.

Disclosures

The authors have nothing to disclose.

Acknowledgements

The authors would like to acknowledge the many contributions to the field that were not cited in this work due to space limitations, as well as the funding agencies that provided this opportunity. The authors acknowledge funding from the Natural Sciences and Engineering Research Council of Canada (NSERC) and the Canadian Institutes of Health Research (CIHR), which supported this work. K.M. is the recipient of a CGS-M scholarship from NSERC and a Killam Doctoral Scholarship from the University of British Columbia. N.S. is the recipient of a Michael Smith Health Research BC Scholar Award.

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

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Maheden, K., Hwang, K., Egilmez, I., Shakiba, N. An Optimized Mouse Embryonic Stem Cell Based Reverse Poly-Transfection Technique for Rapid Exploration of Nucleic Acid Ratios. J. Vis. Exp. (202), e65766, doi:10.3791/65766 (2023).

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