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

Analisi comparativa dei metodi di trasfezione diretta e inversa su 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.

Autore in primo piano: Progressi nei dispositivi genetici sintetici per la manipolazione del destino delle cellule staminali e lo sviluppo della terapia cellulare

Una tecnica di politrasfezione inversa ottimizzata basata su cellule staminali embrionali di topo per una rapida esplorazione dei rapporti degli acidi nucleici

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.

Preparazione di cellule staminali pluripotenti di topo (mPSC) per la trasfezione

Per preparare una piastra da 24 pozzetti ricoperta di gelatina, aggiungere 200 microlitri della soluzione di gelatina allo 0,2% in ciascun pozzetto. Agitare delicatamente la piastra per distribuire uniformemente la soluzione e lasciare che i pozzetti si rivestano a temperatura ambiente. Dopo 15 minuti, utilizzando un aspiratore sottovuoto, rimuovere con cura la gelatina residua dai pozzetti.Aggiungere 0,5 millilitri di terreno NBIL appena preparato a ciascun pozzetto. E posizionare la piastra in un'incubatrice per colture tissutali per preriscaldarla. Aliquotare 30 millilitri di materiale di lavaggio in un tubo conico da 50 millilitri.Aspirare il terreno dalla piastra di coltura tissutale delle mESC e aggiungere la quantità richiesta di terreno di distacco cellulare. Dopo tre-cinque minuti, pipettare su e giù da 15 a 20 volte utilizzando una pipetta P1.000. Osserva le cellule al microscopio.Per garantire una sospensione a cellula singola, trasferire la sospensione cellulare in un tubo da 50 millilitri contenente il mezzo di lavaggio. Centrifugare la sospensione a 300 g per cinque minuti a temperatura ambiente e aspirare il mezzo di lavaggio. Risospendere il pellet in circa 300 microlitri di materiale di lavaggio.

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 ...

Ricerca

Biologia dello sviluppo

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

Trasfezione inversa e diretta di cellule staminali pluripotenti di topo (mPSC)

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

Analisi citometrica a flusso di cellule staminali pluripotenti di topo trasfettate (mPSC)