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

एमपीएससी पर फॉरवर्ड और रिवर्स ट्रांसफेक्शन विधियों का तुलनात्मक विश्लेषण

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.

लेखक स्पॉटलाइट: स्टेम सेल भाग्य हेरफेर और सेलुलर थेरेपी विकास के लिए सिंथेटिक आनुवंशिक उपकरणों में प्रगति

न्यूक्लिक एसिड अनुपात के तेजी से अन्वेषण के लिए एक अनुकूलित माउस भ्रूण स्टेम सेल आधारित रिवर्स पॉली-अभिकर्मक तकनीक

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.

अभिकर्मक के लिए माउस प्लुरिपोटेंट स्टेम सेल (mPSCs) की तैयारी

एक जिलेटिन लेपित 24 अच्छी तरह से थाली तैयार करने के लिए प्रत्येक अच्छी तरह से 0.2% जिलेटिन समाधान के 200 माइक्रोलीटर जोड़ें. धीरे समान रूप से समाधान फैल करने के लिए थाली हिला और कुओं कमरे के तापमान पर कोट करने के लिए अनुमति देते हैं. 15 मिनट के बाद, एक वैक्यूम एस्पिरेटर का उपयोग ध्यान से कुओं से किसी भी बचे हुए जिलेटिन को हटा दें।प्रत्येक कुएं में ताजा तैयार एनबीआईएल मीडिया के 0.5 मिलीलीटर जोड़ें। और प्लेट को टिशू कल्चर इनक्यूबेटर में प्रीवार्म करने के लिए रखें। एक 50 मिलीलीटर शंक्वाकार ट्यूब में धोने के मीडिया के 30 मिलीलीटर विभाज्य।एमईएससी के टिशू कल्चर डिश से मीडिया को एस्पिरेट करें और सेल टुकड़ी माध्यम की आवश्यक मात्रा जोड़ें। तीन से पांच मिनट के बाद, पिपेट ऊपर और नीचे एक पी 1, 000 विंदुक का उपयोग कर 15 से 20 बार. एक खुर्दबीन के तहत कोशिकाओं का निरीक्षण करें.एकल सेल निलंबन सुनिश्चित करने के लिए, सेल निलंबन को वॉश मीडिया युक्त 50 मिलीलीटर ट्यूब में स्थानांतरित करें। कमरे के तापमान पर पांच मिनट के लिए 300 ग्राम पर निलंबन को अपकेंद्रित्र करें और वॉश मीडिया को एस्पिरेट करें। वॉश मीडिया के लगभग 300 माइक्रोलीटर में गोली को फिर से निलंबित करें।

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 ...

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

रिवर्स और माउस प्लुरिपोटेंट स्टेम सेल (mPSCs) के आगे अभिकर्मक

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

ट्रांसफ़ेक्ट माउस प्लुरिपोटेंट स्टेम सेल (mPSCs) का फ्लो साइटोमेट्री विश्लेषण