Retroviral Mediated Gene Transduction: A Gene Transfer Technique to Deliver a Transgene in Cultured Cells Using Engineered Retroviral Vectors
Published: April 30, 2023
Abstract
Source: Jacquier, A. et al. Modeling Charcot-Marie-Tooth Disease In Vitro by Transfecting Mouse Primary Motoneurons. J. Vis. Exp. (2019)
In this video, we demonstrate magnetically-guided transfection of plasmid DNA in primary neuronal cell culture. Magnetofection uses an external magnetic field to guide the delivery of plasmids bound to magnetic nanoparticles into cell cytoplasm.
Protocol
1. Prepare Retroviral Construct Sub-clone the T-cell receptor (TCR) alpha and beta chains, separated by a 2A linker, into an MSCV-based retroviral vector (example, pMIAII or pMIGII). NOTE: The 2A linker allows for the stoichiometric and concordant expression of the alpha and beta TCR chains from a single open reading frame. The vector includes an IRES fluorescent protein cassette (ametrine or GFP) that is used to track transduced cells and transduction efficiency….
開示
The authors have nothing to disclose.
Materials
| DMEM, high glucose + glutamine | Corning Cellgro | 10-013-CV | Dulbecco's Modification of Eagle's Medium with 4.5 g/L glucose, L-glutamine & sodium pyruvate |
| FBS | Atlanta Biological | S11550 | |
| Trypsin-Versene | Lonza | 17-161F | |
| 0.45 μm syringe filter | Thermo Scientific | 194-2545 | |
| Polybrene | Sigma | H9268-10G | Sterile Filtered in dH2O |
| Ciprofloxacin | VWR | AAJ61970-06 | |
| Sodium Pyruvate | Corning Cellgro | 25-000-CI | |
| MEM nonessential Amino Acids | Corning Cellgro | 25-025-CI | |
| HEPES 1 M solution | Corning Cellgro | 25-060-CI | |
| L-glutamine | Corning Cellgro | 25-005-CI | |
| 150 mm tissue culture dishes | Greiner Bio-one | 639160 | |
| Tisue culture-treated 6-well flat plate | Greiner Bio-one | 657160 | |
| 70 μm nylon cell strainers | Falcon | 352350 | |
| Mouse IL-3 | Invitrogen | PMC0033 | |
| Human IL-6 | Invitrogen | PHC0063 | |
| Mouse Stem Cell Factor | Invitrogen | PMC2113L | |
| 10x PBS | Corning Cellgro | 46-D13-CM | |
| HANKS Buffer | Corning Cellgro | 21020147 | |
| BD 10 ml Syringe | BD | 300912 | |
| BD 1 ml Syringe | BD | 309659 | |
| 27 G x 1/2 BD Precision Glide Needle | BD | 305109 | |
| 30 G x 1/2 BD Precision Glide Needle | BD | 305106 |
