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 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.
		<ol>
			<li>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).</li>
			<li>Store solutions at -20 &#176;C for up to 2 years.</li>
		</ol>
		</li>
		<li>In a biosafety cabinet (BSC), add the following to 58.035 mL of DMEM-F12.
		<ol>
			<li>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.</li>
			<li>Filter sterilize the above mixture with a 0.22 &#181;m filter.</li>
		</ol>
		</li>
		<li>Still in the BSC, prepare a 100 mg/mL solution of apotransferrin.
		<ol>
			<li>Add 5 mL of DMEM-F12 to 500 mg of apotransferrin (see Table of Materials). Slowly resuspend and wash the container, avoiding bubbles.</li>
			<li>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.</li>
		</ol>
		</li>
		<li>Add 5 mL of 7.5% BSA fraction V (see Table of Materials) to the solution.</li>
		<li>Mix and aliquot 6.875 mL per tube. Store aliquots at -80 &#176;C for up to 1 year.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Prepare N2B27 basal media.
	<ol>
		<li>Thaw N2 and B27 supplements in a 4 &#176;C fridge for a few hours or overnight.</li>
		<li>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.</li>
		<li>Add 10 mL of B27 and 10 mL of N2 supplement to the media.</li>
		<li>Add 1 mL of 55 mM beta-mercaptoethanol (see Table of Materials). Add 10 mL of 100x glutamax. Mix vigorously by swirling the bottle.</li>
		<li>Aliquot the desired volume per aliquot (typically 100 mL) and store at -80 &#176;C for up to 1 year. Store in use after thawing at 4 &#176;C for up to 3 weeks.</li>
	</ol>
	</li>
	<li>Prepare NBiL media.
	<ol>
		<li>Thaw a 100 mL aliquot of N2B27 basal media from -80 &#176;C in the 4 &#176;C fridge.</li>
		<li>In a BSC, add LIF (see Table of Materials) to a final concentration of 10 &#181;g/L.</li>
		<li>Add CHIR99021 (3 &#181;M final) and PD0325901 (1 &#181;M final) (see Table of Materials). Mix well with a 50 mL serological pipette. Store at 4 &#176;C for up to 2 weeks.</li>
	</ol>
	</li>
	<li>Prepare 0.2% gelatin solution.
	<ol>
		<li>Transfer 500 mL of double-distilled H2O into a 1 L glass bottle.</li>
		<li>Measure out 1 g of gelatin (see Table of Materials) and add it to the water. Mix by shaking the bottle until mostly dissolved.</li>
		<li>Autoclave the gelatin and water mixture at 15 psi, 121 &#176;C for 35 min. Once cooled, filter sterilize it with a 0.22 &#181;m filter in a BSC. Store at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Prepare wash media.
	<ol>
		<li>In a BSC, mix 160 &#181;L of 7.5% BSA per 10 mL of DMEM-F12.</li>
		<li>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 &#176;C.</li>
	</ol>
	</li>
</ol>
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.
<ol>
	<li>Calculate the volume of DNA solution needed per treatment.
	<ol>
		<li>Prepare 500 ng of DNA per well/condition.
		<ol>
			<li>If transfecting a single plasmid with a DNA solution of 100 ng/&#181;L, prepare one tube with 5 &#181;L of DNA.</li>
			<li>If transfecting two plasmids at 100 ng/&#181;L each, prepare two tubes with 2.5 &#181;L in each, one for each plasmid.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>In a BSC, perform the following: For each 500 ng transfection treatment (see Table of Materials), aliquot 25 &#181;L of OptiMEM into a 1.5 mL tube.
	<ol>
		<li>Scale this accordingly - for the example above, prepare two tubes, each with 250 ng of DNA (2.5 &#181;L) and 12.5 &#181;L of OptiMEM.</li>
	</ol>
	</li>
	<li>Add the required volume of DNA for that treatment to the OptiMEM in the tube.</li>
	<li>For each tube with 500 ng DNA and OptiMEM, create a matching tube with another 25 &#181;L of OptiMEM and 1 &#181;L of cationic lipid-based transfection reagent.
	<ol>
		<li>Again, these values must be scaled accordingly. For the above example, prepare two tubes, each with 12.5 &#181;L of OptiMEM and 0.5 &#181;L of transfection reagent. 
		&#8203;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 &#181;L OptiMEM: 2 &#181;L transfection reagent: 50 &#181;L OptiMEM).</li>
	</ol>
	</li>
</ol>
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.
<ol>
	<li>Prepare a 0.2% gelatin-coated cell culture vessel.
	<ol>
		<li>Plan the number of wells needed for the transfection (one well of a 24-well plate per treatment/group).</li>
		<li>Add 200 &#181;L of the 0.2% gelatin solution to each of the desired wells in the 24-well plate.</li>
		<li>Gently shake the plate to evenly spread the solution, ensuring it covers the entire bottom of the well.</li>
		<li>Allow the wells to coat for a minimum of 15 min at room temperature.</li>
		<li>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).</li>
		<li>Add 0.5 mL of NBiL media (step 1.3) to each well and leave the plate in a tissue culture incubator to prewarm.</li>
	</ol>
	</li>
	<li>Passage mPSCs.
	<ol>
		<li>Aliquot 30 mL of wash media (step 1.5) into a 50 mL conical tube.</li>
		<li>Aspirate the old media from the tissue culture dish of mESCs. 
		&#8203;NOTE: Several aspiration techniques are permissible. Whatever technique is chosen, ensure that cells do not remain dry for extended periods (approximately 30 s maximum).</li>
		<li>Add the required amount of commercially available cell detatchment medium (1.5 mL for a 10 cm dish, scale accordingly, see Table of Materials).</li>
		<li>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.</li>
		<li>Transfer the cells in the detatchment medium into the 50 mL tube containing wash media.</li>
		<li>Centrifuge at 300 x g for 5 min at room temperature. Aspirate wash media, ensuring no residual liquid remains in the tube.</li>
		<li>Resuspend the pellet in a small volume of wash media (~300 &#181;L). Count the cell suspension using a hemocytometer to obtain the cell density.</li>
	</ol>
	</li>
</ol>
4. Reverse transfection of mPSCs
<ol>
	<li>Prepare poly-transfection reagents according to step 2.</li>
	<li>Prepare the cell culture vessel and passage mPSCs according to step 3.</li>
	<li>Aliquot 200 &#181;L of NBiL media to 1.5 mL tubes, one for each treatment.</li>
	<li>Add 26 &#181;L 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.</li>
	<li>Based on cell density, transfer the required volume of cells from the 300 &#181;L cell suspension to the 200 &#181;L NBiL tubes to obtain 25,000 cells per tube.</li>
	<li>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.</li>
	<li>Centrifuge at 300 x g for 5 min at room temperature. Pipette out the liquid, leaving approximately 50 &#181;L.</li>
	<li>Resuspend the cell pellet with 100 &#181;L 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.</li>
</ol>
5. Forward transfection of mPSCs
<ol>
	<li>12-18 h prior to transfection, prepare the cell culture vessel and passage mPSCs according to step 3.</li>
	<li>Based on cell density, transfer the required volume of cells from the 300 &#181;L cell mixture to seed 25,000 cells per well. Place the plate in the incubator.</li>
	<li>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.</li>
	<li>At the time of transfection, prepare mPSC poly-transfection reagents according to step 2.</li>
	<li>Add 26 &#181;L 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.</li>
	<li>Add the combined mixture to the wells dropwise. Place the plate in the incubator. Change the media 24 h later.</li>
</ol>
6. Flow cytometry
<ol>
	<li>Aspirate the media from the transfected 24-well plate 48 h after transfection.</li>
	<li>Add 200 &#181;L of trypsin to each well, swirl, and incubate for 30 s at 37 &#176;C. Tap the sides of the plate and add 200 &#181;L of ice-cold FACS buffer (2% FBS in PBS).</li>
	<li>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 &#181;L from each well to the appropriate well on the 96-well plate.</li>
	<li>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.</li>
	<li>Use a multichannel pipette to resuspend the pellets on the 96-well plate in 150 &#181;L 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.</li>
	<li>Run the samples through a flow cytometer to obtain the population distributions of the fluorescent reporters.
	<ol>
		<li>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).</li>
		<li>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.</li>
	</ol>
	</li>
	<li>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.</li>
</ol>

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>

The authors report no conflicts of interest.

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><tbody><tr><td>Accutase&amp;nbsp;</td><td>MilliporeSigma</td><td>SCR005</td><td></td></tr><tr><td>Apotransferrin&amp;nbsp;</td><td>MilliporeSigma</td><td>T1147-500MG</td><td></td></tr><tr><td>B27 supplement&amp;nbsp;</td><td>ThermoFisher Scientific&amp;nbsp;</td><td>17504044</td><td></td></tr><tr><td>Beta-mercaptoethanol</td><td>ThermoFisher Scientific&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</td><td>MilliporeSigma</td><td>G1890</td><td></td></tr><tr><td>GlutaMAX supplement&amp;nbsp;</td><td>ThermoFisher Scientific&amp;nbsp;</td><td>35050061</td><td></td></tr><tr><td>Insulin&amp;nbsp;</td><td>Gibco</td><td>12585-0014</td><td></td></tr><tr><td>Lipofectamine 2000&amp;nbsp;</td><td>Invitrogen</td><td>11668-019</td><td>Transfection reagent</td></tr><tr><td>Neurobasal media</td><td>ThermoFisher Scientific&amp;nbsp;</td><td>21103049</td><td></td></tr><tr><td>OptiMEM&amp;nbsp;</td><td>Invitrogen</td><td>31985-070</td><td></td></tr><tr><td>PD0325901&amp;nbsp;</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&amp;nbsp;</td><td>BioTechne</td><td>8878-LF-500/CF</td><td></td></tr><tr><td>Sodium selenite&amp;nbsp;</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&amp;nbsp;</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.

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

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

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.

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 ...

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952 Views.  University of British Columbia. 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.

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

Loss- and Gain-of-function Approach to Investigate Early Cell Fate Determinants in Preimplantation Mouse Embryos

Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene modification in preimplantation embryos provides a means to study the underlying mechanisms of genes implicated in, for instance, cellular differentiation into the trophectoderm (TE) and the inner cell mass (ICM). Here, we describe a protocol that examines the role of neogenin as an authentic receptor for initial cell fate determination in preimplantation mouse embryos. First, we discuss the experimental manipulations that were used to produce gain and loss of neogenin function by microinjecting neogenin cDNA and shRNA; the effectiveness of this approach was confirmed by a strong correlation between the pair-wise expression levels of either red fluorescent protein (RFP) or green fluorescent protein (GFP) and the immunocytochemical quantification of neogenin expression. Secondly, overexpression of neogenin in preimplantation mouse embryos leads to normal ICM development while neogenin knockdown causes the ICM to develop abnormally, implying that neogenin could be a receptor that relays extracellular cues to drive blastomeres to early cell fates. Given the success of this detailed protocol in investigating the function of a novel embryonic developmental stage-specific receptor, we propose that it has the potential to aid in exploration and identification of other stage-specific genes during embryogenesis.

The goal of this protocol is to describe a loss- and gain-of function method that is applicable to identify neogenin as a stage-specific receptor that leads to trophectoderm and inner cell mass differentiation in preimplantation mouse embryos.

 Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene ...

Reprogramming Mouse Embryonic Fibroblasts with Transcription Factors to Induce a Hemogenic Program

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We first designed a reporter screen using MEFs from human CD34-tTA/TetO-H2BGFP (34/H2BGFP) double transgenic mice. CD34+ cells from these mice label H2B histones with GFP, and cease labeling upon addition of doxycycline (DOX). MEFS were transduced with candidate TFs and then observed for the emergence of GFP+ cells that would indicate the acquisition of a hematopoietic or endothelial cell fate. Starting with 18 candidate TFs, and through a process of combinatorial elimination, we obtained a minimal set of factors that would induce the highest percentage of GFP+ cells. We found that Gata2, Gfi1b, and cFos were necessary and the addition of Etv6 provided the optimal induction. A series of gene expression analyses done at different time points during the reprogramming process revealed that these cells appeared to go through a precursor cell that underwent an endothelial to hematopoietic transition (EHT). As such, this reprogramming process mimics developmental hematopoiesis "in a dish," allowing study of hematopoiesis in vitro and a platform to identify the mechanisms that underlie this specification. This methodology also provides a framework for translation of this work to the human system in the hopes of generating an alternative source of patient-specific hematopoietic stem cells (HSCs) for a number of applications in the treatment and study of hematologic diseases.

The protocol described here details the induction of a hemogenic program in mouse embryonic fibroblasts via overexpression of a minimal set of transcription factors. This technology may be translated to the human system to provide platforms for future study of hematopoiesis, hematologic disease, and hematopoietic stem cell transplant.

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We ...

Identification of Homologous Recombination Events in Mouse Embryonic Stem Cells Using Southern Blotting and Polymerase Chain Reaction

Relative to the issues of off-target effects and the difficulty of inserting a long DNA fragment in the application of designer nucleases for genome editing, embryonic stem (ES) cell-based gene-targeting technology does not have these shortcomings and is widely used to modify animal/mouse genome ranging from large deletions/insertions to single nucleotide substitutions. Notably, identifying the relatively few homologous recombination (HR) events necessary to obtain desired ES clones is a key step, which demands accurate and reliable methods. Southern blotting and/or conventional PCR are often utilized for this purpose. Here, we describe the detailed procedures of using those two methods to identify HR events that occurred in mouse ES cells in which the endogenous Myh9 gene is intended to be disrupted and replaced by cDNAs encoding other nonmuscle myosin heavy chain IIs (NMHC IIs). The whole procedure of Southern blotting includes the construction of targeting vector(s), electroporation, drug selection, the expansion and storage of ES cells/clones, the preparation, digestion, and blotting of genomic DNA (gDNA), the hybridization and washing of probe(s), and a final step of autoradiography on the X-ray films. PCR can be performed directly with prepared and diluted gDNA. To obtain ideal results, the probes and restriction enzyme (RE) cutting sites for Southern blotting and the primers for PCR should be carefully planned. Though the execution of Southern blotting is time-consuming and labor-intensive and PCR results have false positives, the correct identification by Southern blotting and the rapid screening by PCR allow the sole or combined application of these methods described in this paper to be widely used and consulted by most labs in the identification of genotypes of ES cells and genetically modified animals.

Here, we present a detailed protocol for identifying homologous recombination events that occurred in mouse embryonic stem cells using Southern blotting and/or PCR. This method is exemplified by the generation of nonmuscle myosin II genetic replacement mouse models using traditional embryonic stem cell-based homologous recombination-mediated targeting technology.

Relative to the issues of off-target effects and the difficulty of inserting a long DNA fragment in the application of designer nucleases for genome ...

Genetics

Lentiviral Vector Platform for the Efficient Delivery of Epigenome-editing Tools into Human Induced Pluripotent Stem Cell-derived Disease Models

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the differentiation of hiPSCs derived from a patient with the triplication of the alpha-synuclein gene (SNCA) locus into Parkinson&#8217;s disease (PD)-relevant dopaminergic neuronal populations. Accumulating evidence has shown that high levels of SNCA are causative for the development of PD. Recognizing the unmet need to establish novel therapeutic approaches for PD, especially those targeting the regulation of SNCA expression, we recently developed a CRISPR/dCas9-DNA-methylation-based system to epigenetically modulate SNCA transcription by enriching methylation levels at the SNCA intron 1 regulatory region. To deliver the system, consisting of a dead (deactivated) version of Cas9 (dCas9) fused with the catalytic domain of the DNA methyltransferase enzyme 3A (DNMT3A), a lentiviral vector is used. This system is applied to cells with the triplication of the SNCA locus and reduces the SNCA-mRNA and protein levels by about 30% through the targeted DNA methylation of SNCA intron 1. The fine-tuned downregulation of the SNCA levels rescues disease-related cellular phenotypes. In the current protocol, we aim to describe a step-by-step procedure for differentiating hiPSCs into neural progenitor cells (NPCs) and the establishment and validation of pyrosequencing assays for the evaluation of the methylation profile in the SNCA intron 1. To outline in more detail the lentivirus-CRISPR/dCas9 system used in these experiments, this protocol describes how to produce, purify, and concentrate lentiviral vectors and to highlight their suitability for epigenome- and genome-editing applications using hiPSCs and NPCs. The protocol is easily adaptable and can be used to produce high titer lentiviruses for in vitro and in vivo applications.

Targeted DNA epigenome editing represents a powerful therapeutic approach. This protocol describes the production, purification, and concentration of all-in-one lentiviral vectors harboring the CRISPR-dCas9-DNMT3A transgene for epigenome-editing applications in human induced pluripotent stem cell (hiPSC)-derived neurons.

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the ...

In ovo Electroporation of miRNA-based Plasmids in the Developing Neural Tube and Assessment of Phenotypes by DiI Injection in Open-book Preparations

Commissural dI1 neurons have been extensively studied to elucidate the mechanisms underlying axon guidance during development1,2. These neurons are located in the dorsal spinal cord and send their axons along stereotyped trajectories. Commissural axons initially project ventrally towards and then across the floorplate. After crossing the midline, these axons make a sharp rostral turn and project longitudinally towards the brain. Each of these steps is regulated by the coordinated activities of attractive and repulsive guidance cues. The correct interpretation of these cues is crucial to the guidance of axons along their demarcated pathway. Thus, the physiological contribution of a particular molecule to commissural axon guidance is ideally investigated in the context of the living embryo. Accordingly, gene knockdown in vivo must be precisely controlled in order to carefully distinguish axon guidance activities of genes that may play multiple roles during development.
Here, we describe a method to knockdown gene expression in the chicken neural tube in a cell type-specific, traceable manner. We use novel plasmid vectors3 harboring cell type-specific promoters/enhancers that drive the expression of a fluorescent protein marker, followed directly by a miR30-RNAi transcript4 (located within the 3'-UTR of the cDNA encoding the fluorescent protein) (Figure 1). When electroporated into the developing neural tube, these vectors elicit efficient downregulation of gene expression and express bright fluorescent marker proteins to enable direct tracing of the cells experiencing knockdown3. Mixing different RNAi vectors prior to electroporation allows the simultaneous knockdown of two or more genes in independent regions of the spinal cord. This permits complex cellular and molecular interactions to be examined during development, in a manner that is fast, simple, precise and inexpensive. In combination with DiI tracing of commissural axon trajectories in open-book preparations5, this method is a useful tool for in vivo studies of the cellular and molecular mechanisms of commissural axon growth and guidance. In principle, any promoter/enhancer could be used, potentially making the technique more widely applicable for in vivo studies of gene function during development6.
This video first demonstrates how to handle and window eggs, the injection of DNA plasmids into the neural tube and the electroporation procedure. To investigate commissural axon guidance, the spinal cord is removed from the embryo as an open-book preparation, fixed, and injected with DiI to enable axon pathways to be traced. The spinal cord is mounted between coverslips and visualized using confocal microscopy.

In ovo Electroporation of miRNA-based Plasmids in the Developing Neural Tube and Assessment of Phenotypes by DiI Injection in Open-book Preparations

A method by which gene expression in the neural tube can be downregulated in a cell type-specific, traceable manner is described. We demonstrate how in ovo electroporation of microRNA-based plasmids that elicit spatiotemporally controlled RNA interference can be used to investigate commissural axon guidance in the developing neural tube.

Commissural dI1 neurons have been extensively studied to elucidate the mechanisms underlying axon guidance during development1,2. These neurons are ...

Neuroscience

CRISPR/Cas9-Mediated Highly Efficient Gene Targeting in Embryonic Stem Cells for Developing Gene-Manipulated Mouse Models

The CRISPR/Cas9 system has made it possible to develop genetically modified mice by direct genome editing using fertilized zygotes. However, although the efficiency in developing gene-knockout mice by inducing small indel mutation would be sufficient enough, the efficiency of embryo genome editing for making large-size DNA knock-in (KI) is still low. Therefore, in contrast to the direct KI method in embryos, gene targeting using embryonic stem cells (ESCs) followed by embryo injection to develop chimera mice still has several advantages (e.g., high throughput targeting in vitro, multi-allele manipulation, and Cre and flox gene manipulation can be carried out in a short period). In addition, strains with difficult-to-handle embryos in vitro, such as BALB/c, can also be used for ESC targeting. This protocol describes the optimized method for large-size DNA (several kb) KI in ESCs by applying CRISPR/Cas9-mediated genome editing followed by chimera mice production to develop gene-manipulated mouse models.

Here we present a protocol for developing genetically modified mouse models using embryonic stem cells, especially for large DNA knock-in (KI). This protocol is tuned up using CRISPR/Cas9 genome editing, resulting in significantly improved KI efficiency compared with the conventional homologous recombination-mediated linearized DNA targeting method.

The CRISPR/Cas9 system has made it possible to develop genetically modified mice by direct genome editing using fertilized zygotes. However, although the ...

Oct4GiP Reporter Assay to Study Genes that Regulate Mouse Embryonic Stem Cell Maintenance and Self-renewal

Pluripotency and self-renewal are two defining characteristics of embryonic stem cells (ES cells). Understanding the underlying molecular mechanism will greatly facilitate the use of ES cells for developmental biology studies, disease modeling, drug discovery, and regenerative medicine (reviewed in 1,2).
To expedite the identification and characterization of novel regulators of ES cell maintenance and self-renewal, we developed a fluorescence reporter-based assay to quantitatively measure the self-renewal status in mouse ES cells using the Oct4GiP cells 3. The Oct4GiP cells express the green fluorescent protein (GFP) under the control of the Oct4 gene promoter region 4,5. Oct4 is required for ES cell self-renewal, and is highly expressed in ES cells and quickly down-regulated during differentiation 6,7. As a result, GFP expression and fluorescence in the reporter cells correlates faithfully with the ES cell identity 5, and fluorescence-activated cell sorting (FACS) analysis can be used to closely monitor the self-renewal status of the cells at the single cell level 3,8.
Coupled with RNAi, the Oct4GiP reporter assay can be used to quickly identify and study regulators of ES cell maintenance and self-renewal 3,8. Compared to other methods for assaying self-renewal, it is more convenient, sensitive, quantitative, and of lower cost. It can be carried out in 96- or 384-well plates for large-scale studies such as high-throughput screens or genetic epistasis analysis. Finally, by using other lineage-specific reporter ES cell lines, the assay we describe here can also be modified to study fate specification during ES cell differentiation.

We describe a fluorescence reporter assay to quickly identify and characterize genes that regulate mouse embryonic stem cell maintenance and self-renewal.

Pluripotency and self-renewal are two defining characteristics of embryonic stem cells (ES cells). Understanding the underlying molecular mechanism will ...

Biology

Rapid Development of Cell State Identification Circuits with Poly-Transfection

Mammalian genetic circuits have demonstrated the potential to sense and treat a wide range of disease states, but optimization of the levels of circuit components remains challenging and labor-intensive. To accelerate this process, our lab developed poly-transfection, a high-throughput extension of traditional mammalian transfection. In poly-transfection, each cell in the transfected population essentially performs a different experiment, testing the behavior of the circuit at different DNA copy numbers and allowing users to analyze a large number of stoichiometries in a single-pot reaction. So far, poly-transfections that optimize ratios of three-component circuits in a single well of cells have been demonstrated; in principle, the same method can be used for the development of even larger circuits. Poly-transfection results can be easily applied to find optimal ratios of DNA to co-transfect for transient circuits or to choose expression levels for circuit components for the generation of stable cell lines.
Here, we demonstrate the use of poly-transfection to optimize a three-component circuit. The protocol begins with experimental design principles and explains how poly-transfection builds upon&#160;traditional co-transfection methods. Next, poly-transfection of cells is carried out and followed by flow cytometry a few days later. Finally, the data is analyzed by examining slices of the single-cell flow cytometry data that correspond to subsets of cells with certain component ratios. In the lab, poly-transfection has been used to optimize cell classifiers, feedback and feedforward controllers, bistable motifs, and many more. This simple but powerful method speeds up design cycles for complex genetic circuits in mammalian cells.

Complex genetic circuits are time-consuming to design, test, and optimize. To facilitate this process, mammalian cells are transfected in a way that allows the testing of multiple stoichiometries of circuit components in a single well. This protocol outlines the steps for experimental planning, transfection, and data analysis.

Mammalian genetic circuits have demonstrated the potential to sense and treat a wide range of disease states, but optimization of the levels of circuit ...

Bioengineering

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different lineages. A single cell transcriptome assay can be a new approach for dissecting individual variation. We have developed the single cell qRT-PCR method, and confirmed that this method works well in several gene expression profiles. In single cell level, each human embryonic stem cell, sorted by OCT4::EGFP positive cells, has high expression in OCT4, but a different level of NANOG expression. Our single cell gene expression assay should be useful to interrogate population heterogeneities.

Single cell gene expression assay is needed for understanding stem cell heterogeneities.

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

Zinc-finger Nuclease Enhanced Gene Targeting in Human Embryonic Stem Cells

One major limitation with current human embryonic stem cell (ESC) differentiation protocols is the generation of heterogeneous cell populations.&#160;These cultures contain the cells of interest, but are also contaminated with undifferentiated ESCs, non-neural derivatives and other neuronal subtypes.&#160; This limits their use in in vitro and in vivo applications, such as in vitro modeling for drug discovery or cell replacement therapy. To help overcome this, reporter cell lines, which offer a means to visualize, track and isolate cells of interest, can be engineered.&#160;However, to achieve this in human embryonic stem cells via conventional homologous recombination is extremely inefficient.&#160;This protocol describes targeting of the Pituitary homeobox 3 (PITX3) locus in human embryonic stem cells using custom designed zinc-finger nucleases, which introduce site-specific double-strand DNA breaks, together with a PITX3-EGFP-specific DNA donor vector. Following the generation of the PITX3 reporter cell line, it can then be differentiated using published protocols for use in studies such as in vitro Parkinson&#8217;s disease modeling or cell replacement therapy.

Reporter cell lines offer a means to visualize, track and isolate cells of interest from heterogeneous populations.&#160;However, gene-targeting using conventional homologous recombination in human embryonic stem cells is extremely inefficient. Herein, we describe targeting CNS midbrain specific transcription factor PITX3 locus with EGFP using zinc-finger nuclease enhanced homologous recombination.

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

Summary

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Chapters in this video

Loss- and Gain-of-function Approach to Investigate Early Cell Fate Determinants in Preimplantation Mouse Embryos

Reprogramming Mouse Embryonic Fibroblasts with Transcription Factors to Induce a Hemogenic Program

Identification of Homologous Recombination Events in Mouse Embryonic Stem Cells Using Southern Blotting and Polymerase Chain Reaction

Lentiviral Vector Platform for the Efficient Delivery of Epigenome-editing Tools into Human Induced Pluripotent Stem Cell-derived Disease Models

In ovo Electroporation of miRNA-based Plasmids in the Developing Neural Tube and Assessment of Phenotypes by DiI Injection in Open-book Preparations

CRISPR/Cas9-Mediated Highly Efficient Gene Targeting in Embryonic Stem Cells for Developing Gene-Manipulated Mouse Models

Oct4GiP Reporter Assay to Study Genes that Regulate Mouse Embryonic Stem Cell Maintenance and Self-renewal

Rapid Development of Cell State Identification Circuits with Poly-Transfection

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Zinc-finger Nuclease Enhanced Gene Targeting in Human Embryonic Stem Cells