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

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記事を引用
Retroviral Mediated Gene Transduction: A Gene Transfer Technique to Deliver a Transgene in Cultured Cells Using Engineered Retroviral Vectors. J. Vis. Exp. (Pending Publication), e20998, doi: (2023).

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