preparation of mouse pluripotent stem cells &#40;mpscs&#41; for transfection

Analyse comparative des méthodes de transfection directe et inverse sur les mPSC

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.

Pleins feux sur l’auteur : Progrès dans les dispositifs génétiques synthétiques pour la manipulation du destin des cellules souches et le développement de thérapies cellulaires

Technique de poly-transfection inverse optimisée à base de cellules souches embryonnaires de souris pour l’exploration rapide des ratios d’acides nucléiques

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.

Préparation de cellules souches pluripotentes de souris (mPSC) pour la transfection

Pour préparer une plaque de 24 puits enrobée de gélatine, ajoutez 200 microlitres de la solution de gélatine à 0,2 % dans chaque puits. Secouez doucement la plaque pour répartir uniformément la solution et laissez les puits s’enrober à température ambiante. Après 15 minutes, à l’aide d’un aspirateur sous vide, retirez soigneusement les restes de gélatine des puits.Ajouter 0,5 millilitre de milieu NBIL fraîchement préparé dans chaque puits. Et placez la plaque dans un incubateur de culture tissulaire pour la préchauffer. Aliquote 30 millilitres de produit de lavage dans un tube conique de 50 millilitres.Aspirer le milieu de la boîte de culture tissulaire des CSEm et ajouter la quantité requise de milieu de détachement cellulaire. Après trois à cinq minutes, pipeter de haut en bas 15 à 20 fois à l’aide d’une pipette P1 000. Observez les cellules au microscope.Pour garantir une suspension à cellule unique, transférez la suspension cellulaire dans un tube de 50 millilitres contenant du produit de lavage. Centrifuger la suspension à 300 G pendant cinq minutes à température ambiante et aspirer le produit de lavage. Remettre le granulé en suspension dans environ 300 microlitres de produit de lavage.

Research

JoVE Journal

Developmental Biology

Preparation of Mouse Pluripotent Stem Cells &#40;mPSCs&#41; for Transfection

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 ...

Recherche

Biologie du développement

Reverse and Forward Transfection of Mouse Pluripotent Stem Cells &#40;mPSCs&#41;

Transfection inverse et directe de cellules souches pluripotentes de souris (mPSC)

Flow Cytometry Analysis of Transfected Mouse Pluripotent Stem Cells &#40;mPSCs&#41;

Analyse par cytométrie en flux de cellules souches pluripotentes de souris transfectées (mPSC)