Encyclopedia of Experiments: Immunology
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Transcript
Take a microwell plate coated with recombinant fibronectin fragments and add activated murine CD8-positive T cells.
Introduce retroviral vectors containing a transgenic RNA that encodes a chimeric antigen receptor or CAR.
Cover the plate and seal it in a bag to avoid contamination. Centrifuge the plate at a low speed to facilitate virus-cell interaction.
Fibronectin binds to the virus and the cell, colocalizing them to enhance virus-mediated gene transfer.
Post-centrifugation, remove the sealed bag and incubate the plate under physiological conditions.
The envelope of the viral vector fuses with the cell membrane, releasing the viral RNA and enzymes.
The viral enzymes reverse-transcribe the released RNA and integrate the resulting DNA into the host genome, enabling the cells to express surface-bound CARs.
The CAR comprises an antigen-binding domain specific to the target antigen and signaling domains for T cell activation.
The antigen-specific CAR T cells are ready for downstream assays.
