Retroviral Mediated Gene Transduction: A Gene Transfer Technique to Deliver a Transgene in Cultured Cells Using Engineered Retroviral Vectors

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

1. 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.
2. Isolate Plasmid DNA using a commercially obtained plasma prep kit or a similar product. Use a working concentration of 1-2 μg μl^-....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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