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

mPSC에 대한 Forward and Reverse Transfection Method의 비교 분석

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.

저자 스포트라이트: Advancements in Synthetic Genetic Devices for Stem Cell Fate Manipulation and Cellular Therapy Development

핵산 비율의 신속한 탐색을 위해 최적화된 마우스 배아 줄기 세포 기반 역 Poly-transfection 기술

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.

형질주입을 위한 마우스 만능줄기세포(mPSC) 준비

젤라틴 코팅 된 24 웰 플레이트를 준비하려면 각 웰에 200 % 젤라틴 용액 0.2 마이크로 리터를 첨가하십시오. 접시를 부드럽게 흔들어 용액을 고르게 퍼뜨리고 웰이 실온에서 코팅되도록 합니다. 15분 후 진공 흡입기를 사용하여 우물에서 남은 젤라틴을 조심스럽게 제거합니다.각 웰에 갓 준비한 NBIL 배지 0.5ml를 추가합니다. 그리고 플레이트를 조직 배양 인큐베이터에 넣어 예열합니다. 30밀리리터의 세척 매체를 50밀리리터 원뿔형 튜브에 분취합니다.mESC의 조직 배양 접시에서 배지를 흡인하고 필요한 양의 세포 분리 배지를 추가합니다. 3-5분 후, P1, 000 피펫을 사용하여 15-20회 위아래로 피펫팅합니다. 현미경으로 세포를 관찰하십시오.단일 셀 현탁액을 보장하려면 셀 현탁액을 세척 매체가 들어 있는 50mL 튜브로 옮깁니다. 실온에서 300G에서 5분 동안 현탁액을 원심분리하고 세척제를 흡입합니다. 펠릿을 약 300마이크로리터의 세척 매체에 재현탁시킵니다.

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 ...

연구

발생학

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

마우스 만능 줄기 세포(mPSC)의 역방향 및 순방향 형질주입

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

형질주입된 마우스 만능 줄기세포(mPSC)의 유세포 분석