需要订阅 JoVE 才能查看此. 登录或开始免费试用。
An Optimized Mouse Embryonic Stem Cell Based Reverse Poly-Transfection Technique for Rapid Exploration of Nucleic Acid Ratios
In This Article
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 an....
Protocol
1. Preparation of reagents for mPSC culture
- Prepare N2 supplement.
- In non-sterile conditions, prepare the following stock solutions (step 1.1.1.1) in a chemical safety fume hood. Add the solid powder of each chemical to a pre-weighed 50 mL conical tube. Weigh each tube after adding the powder and add an appropriate amount of solvent to achieve the concentrations below. For one batch of media, prepare the minimum amount listed.
NOTE: The following reagents are hazardous and.......
- 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.
Representative Results
Both forward and reverse transfections rely on the interaction between the cell membrane and incoming transfection reagent-DNA complexes, allowing the delivery of NA to the recipient cells. Where these techniques differ is the state of the cell upon delivery - while DNA is typically delivered to adherent cell monolayers in traditional forward transfection, reverse transfection instead relies on having the reagent-DNA complex meet the cells while in a single-cell suspension. This difference can be particularly crucial in .......
Discussion
A key reason for the widespread adoption of transfection protocols is their reproducibility and accessibility; however, these protocols do require optimization across experimental contexts. Not mentioned above is the standard testing required when attempting to transfect a new cell line for the first time. First, the choice of transfection reagent is key, as commercially available reagents are not one-size-fits-all and will vary in the efficiency of NA delivery viability across cell types. Additionally, finding the ideal.......
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
| Name | Company | Catalog Number | Comments |
| 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 |
References
- Shakiba, N., Jones, R. D., Weiss, R., Del Vecchio, D. Context-aware synthetic biology by controller design: Engineering the mammalian cell. Cell Systems. 12 (6), 561-592 (2021).
- Fus-Kujawa, A., et al.
This article has been published
Video Coming Soon
关于 JoVE
版权所属 © 2025 MyJoVE 公司版权所有，本公司不涉及任何医疗业务和医疗服务。