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.&#160;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.

1. Preparation of reagents for mPSC culture
<ol>
	<li>Prepare N2 supplement.
	<ol>
		<li>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...

Comparative Analysis of Forward and Reverse Transfection Methods on mPSCs

<ol>
	<li>Shakiba, N., Jones, R. D., Weiss, R., Del Vecchio, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Context-aware+synthetic+biology+by+controller+design:+Engineering+the+mammalian+cell.">Context-aware synthetic biology by controller design: Engineering the mammalian cell.</a> Cell Systems. 12 (6), 561-592 (2021).</li><li>Fus-Kujawa, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+overview+of+methods+and+tools+for+transfection+of+eukaryotic+cells+in+vitro.">An overview of methods and tools for transfection of eukaryotic cells in vitro.</a> Frontiers in Bioengineering and Biotechnology. 9, (2021).</li><li>FAU, K. T. K., Eberwine, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+cell+transfection:+the+present+and+the+future.">Mammalian cell transfection: the present and the future.</a> Analytical and Bioanalytical Chemistry. 397 (8), 3173-3178 (2010).</li><li>Tamm, C., Galit&#243;, S. P., Anner&#233;n, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+study+of+protocols+for+mouse+embryonic+stem+cell+culturing.">A comparative study of protocols for mouse embryonic stem cell culturing.</a> PLoS One. 8 (12), 81156 (2013).</li><li>Morgani, S., Nichols, J., Hadjantonakis, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+many+faces+of+pluripotency:+in+vitro+adaptations+of+a+continuum+of+in+vivo+states.">The many faces of pluripotency: in vitro adaptations of a continuum of in vivo states.</a> BMC Developmental Biology. 17 (1), (2017).</li><li>Mulas, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defined+conditions+for+propagation+and+manipulation+of+mouse+embryonic+stem+cells.">Defined conditions for propagation and manipulation of mouse embryonic stem cells.</a> Development. 146 (6), 173146 (2019).</li><li>Tamm, C., Kadekar, S., Pijuan-Galit&#243;, S., Anner&#233;n, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+and+efficient+transfection+of+mouse+embryonic+stem+cells+using+non-viral+reagents.">Fast and efficient transfection of mouse embryonic stem cells using non-viral reagents.</a> Stem Cell Reviews and Reports. 12 (5), 584-591 (2016).</li><li>Chong, Z. X., Yeap, S. K., Ho, W. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfection+types,+methods+and+strategies:+A+technical+review.">Transfection types, methods and strategies: A technical review.</a> PeerJ. 9, 11165 (2021).</li><li>Gam, J. J., DiAndreth, B., Jones, R. D., Huh, J., Weiss, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+&amp;#34;poly-transfection&amp;#34;+method+for+rapid,+one-pot+characterization+and+optimization+of+genetic+systems.">A &amp;#34;poly-transfection&amp;#34; method for rapid, one-pot characterization and optimization of genetic systems.</a> Nucleic Acids Research. 47 (18), 106 (2019).</li><li>Jones, R. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+endoribonuclease-based+feedforward+controller+for+decoupling+resource-limited+genetic+modules+in+mammalian+cells.">An endoribonuclease-based feedforward controller for decoupling resource-limited genetic modules in mammalian cells.</a> Nature Communications. 11 (1), 5690 (2020).</li><li>Wauford, N., Jones, R., Van De Mark, C., Weiss, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+development+of+cell+state+identification+circuits+with+poly-transfection.">Rapid development of cell state identification circuits with poly-transfection.</a> Journal of Visualized Experiments. (192), e64793 (2023).</li><li>Hori, Y., Kantak, C., Murray, R. M., Abate, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-free+extract+based+optimization+of+biomolecular+circuits+with+droplet+microfluidics.">Cell-free extract based optimization of biomolecular circuits with droplet microfluidics.</a> Lab on a Chip. 17 (18), 3037-3042 (2017).</li><li>Teague, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoflow:+A+python+toolbox+for+flow+cytometry.">Cytoflow: A python toolbox for flow cytometry.</a> bioRxiv. , (2023).</li><li>Qin, J. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+comparison+of+constitutive+promoters+and+the+doxycycline-inducible+promoter.">Systematic comparison of constitutive promoters and the doxycycline-inducible promoter.</a> PLoS One. 5 (5), 0010611 (2010).</li></ol>

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...

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&#160;(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 &#34;poly-transfection,&#34; 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Accutase&#160;</td>
			<td>MilliporeSigma</td>
			<td>SCR005</td>
			<td></td>
		</tr>
		<tr>
			<td>Apotransferrin&#160;</td>
			<td>MilliporeSigma</td>
			<td>T1147-500MG</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 supplement&#160;</td>
			<td>ThermoFisher Scientific&#160;</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>Beta-mercaptoethanol</td>
			<td>ThermoFisher Scientific&#160;</td>
			<td>21985023</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA fraction V (7.5%)</td>
			<td>Gibco</td>
			<td>15260-037</td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR99021&#160;</td>
			<td>MilliporeSigma</td>
			<td>SML1046-25MG</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM-F12</td>
			<td>MilliporeSigma</td>
			<td>D6421-24X500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometry standardization beads</td>
			<td>Spherotech</td>
			<td>URCP-38-2K</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin&#160;</td>
			<td>MilliporeSigma</td>
			<td>G1890</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX supplement&#160;</td>
			<td>ThermoFisher Scientific&#160;</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin&#160;</td>
			<td>Gibco</td>
			<td>12585-0014</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 2000&#160;</td>
			<td>Invitrogen</td>
			<td>11668-019</td>
			<td>Transfection reagent</td>
		</tr>
		<tr>
			<td>Neurobasal media</td>
			<td>ThermoFisher Scientific&#160;</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>OptiMEM&#160;</td>
			<td>Invitrogen</td>
			<td>31985-070</td>
			<td></td>
		</tr>
		<tr>
			<td>PD0325901&#160;</td>
			<td>MilliporeSigma</td>
			<td>PZ0162-25MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Progesterone</td>
			<td>MilliporeSigma</td>
			<td>P8783</td>
			<td>Chemical hazard - consult local safety guidelines, ensure proper PPE is worn, and work with the solid powder form only in a chemical fume hood</td>
		</tr>
		<tr>
			<td>Putrescine</td>
			<td>MilliporeSigma</td>
			<td>P6780</td>
			<td>Chemical hazard - consult local safety guidelines, ensure proper PPE is worn, and work with the solid powder form only in a chemical fume hood</td>
		</tr>
		<tr>
			<td>Recombinant mLIF&#160;</td>
			<td>BioTechne</td>
			<td>8878-LF-500/CF</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium selenite&#160;</td>
			<td>MilliporeSigma</td>
			<td>S5261-25G</td>
			<td>Chemical hazard - consult local safety guidelines, ensure proper PPE is worn, and work with the solid powder form only in a chemical fume hood</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>ThermoFisher Scientific&#160;</td>
			<td>25200056</td>
			<td></td>
		</tr>
	</tbody>
</table>

an optimized mouse embryonic stem cell based reverse poly-transfection technique for rapid exploration of nucleic acid ratios

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.

Author Spotlight: Advancements in Synthetic Genetic Devices for Stem Cell Fate Manipulation and Cellular Therapy Development

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

Our work generally focuses on using synthetic genetic devices to manipulate stem cell fate and behavior. By controlling cellular states, we aim to advance the development of next generation cellular therapies. Recently, a deeper understanding of the relationship between the dosage of a fate determining gene and the cell's lineage has been established.This was facilitated by the development of genetic controllers that are able to titrate a given gene precisely. Classic molecular biology techniques such as transfection or lentiviral transduction for simple gene over expression will always be a mainstay. However, our field has recently adopted synthetic devices that incorporate linear and non-linear feedback in order to obtain more complex outputs from delivered DNA.Gene dosage has a key role in determining the outcome of gene expression. When delivering multiple exogenous genes, a key experimental factor is how much of each gene will give the optimal or desired outcome. Our protocol allows researchers to rapidly scan across dosages in a single treatment, looking for the desired ratio of DNA devices.

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 ...

Watch this Scientific Journal Video about An Optimized Mouse Embryonic Stem Cell Based Reverse Poly-Transfection Technique for Rapid Exploration of Nucleic Acid Ratios at JoVE.com

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.

Due to its relative simplicity and ease of use, transient transfection of mammalian cell lines with nucleic acids has become a mainstay in biomedical ...