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Immunology JoVE (Open Access) Citable · DOI

Retroviral Transduction of T-cell Receptors in Mouse T-cells

DOI: 10.3791/2307-v
What you'll learn
  • ✓ Produce antigen-specific mouse T-cells via retroviral transduction
  • ✓ Isolate and activate primary T-cells from mouse spleen
  • ✓ Evaluate transduction efficiency and assess TCR expression
  • ✓ Expand transduced T-cell populations for downstream applications
Protocol

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

Difficulty
advanced
Total time
~5-7 days (including viral production, T-cell isolation, activation, infection, and expansion phases)
Model organism
Mouse (strain not specified)
Biosafety
BSL-2

Steps

1
Perform retroviral transfection and prepare producer cells

Transfect packaging cells with retroviral vectors containing TCR genes to generate replication-incompetent viral particles for downstream T-cell transduction.

▶ 01:37
2
Prepare single-cell suspension from mouse spleen

Dissociate mouse spleen tissue into a single-cell suspension using mechanical and enzymatic digestion for subsequent T-cell isolation.

▶ 03:55
3
Isolate and activate primary mouse T-cells

Select T-cells from spleen suspension using magnetic beads or flow cytometry, then activate with anti-CD3/CD28 stimulation to enhance transduction competency.

▶ 04:51
4
Infect activated T-cells with retroviral particles

Expose activated T-cells to retroviral supernatant under conditions promoting viral integration and TCR gene transfer into target cells.

▶ 06:27
5
Expand transduced cells and assess integration efficiency

Culture transduced T-cells in expansion medium and measure TCR expression levels using flow cytometry or quantitative PCR to confirm successful transduction.

▶ 08:21
6
Validate retroviral TCR expression on T-cell surface

Confirm functional expression of transduced TCR on expanded T-cell population using flow cytometry with TCR-specific antibodies.

▶ 08:55
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