reverse and forward transfection of mouse pluripotent stem cells &#40;mpscs&#41;

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

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

Pour commencer, aliquote 200 microlitres de média NBIL dans des tubes de 1,5 millilitre. Ajoutez 26 microlitres du mélange optimal de réactifs de transfection au mélange optimal d’ADN. Appliquez vigoureusement les réactifs environ 10 fois.Transférez le volume requis de suspensions mPSC dans les 200 microlitres de tubes NBIL. Ajoutez ensuite la lipofectamine 2000 et le mélange d’ADN dans les tubes et mélangez bien par pipetage. Après cinq minutes, centrifuger le mélange à 300 G pendant cinq minutes et retirer le surnageant en laissant environ 50 microlitres dans le tube.Remettre la pastille en suspension dans 100 microlitres de média NBIL. Transférez la suspension cellulaire dans une plaque à 24 puits recouverte de gélatine et placez la plaque dans l’incubateur. Transférez le volume requis de suspension mPSC dans 25 000 cellules par puits d’une plaque de 24 puits recouverte de gélatine et placez la plaque dans l’incubateur.Une heure avant la transfection. Remplacez le média des puits par 0,5 millilitre de média NBIL. Après l’incubation, ajoutez goutte à goutte la lipofectamine 2000 et le mélange d’ADN dans les puits.Placez la plaque dans l’incubateur pendant 48 heures.

reverse-and-forward-transfection-of-mouse-pluripotent-stem-cells-mpscs

Research

JoVE Journal

Developmental Biology

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

71 vues. Pour commencer, aliquote 200 microlitres de média NBIL dans des tubes de 1,5 millilitre. Ajoutez 26 microlitres du mélange optimal de réactifs de transfection au mélange optimal d’ADN. Appliquez vigoureusement les réactifs environ 10 fois.Transférez le volume requis de suspensions mPSC dans les 200 microlitres de tubes NBIL. Ajoutez ensuite la lipofectamine 2000 et le mélange d’ADN dans les tubes et mélangez bien par pipetage. Après cinq minutes, centrifuger le mélange à...

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

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.

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 ...