Home › Cell Biology › Packaging HIV- or FIV-based Lentivector Expression Constructs & Transduction of VSV-G Pseudotyped Viral Particles
Cell Biology JoVE (Open Access) Citable · DOI

Packaging HIV- or FIV-based Lentivector Expression Constructs & Transduction of VSV-G Pseudotyped Viral Particles

DOI: 10.3791/3171-v
What you'll learn
  • ✓ Package lentiviral expression constructs into pseudotyped viral particles
  • ✓ Perform 293TN cell transfection with packaging plasmids
  • ✓ Concentrate viral supernatant and determine viral titer
  • ✓ Transduce mammalian target cells with lentiviral particles
Protocol

Lentiviral expression vectors are the most effective vehicles for stably expressing different effector molecules or reporter constructs in dividing and non-dividing mammalian cells and whole organisms. Here we provide a protocol on how to package lentivector expression constructs in pseudoviral particles and to transduce target cells using the pseudoviral particles.

Difficulty
advanced
Total time
~3–4 days (transfection, viral production, concentration, titering)
Model organism
HEK293T, HT1080
Biosafety
BSL-2

Steps

1
Understand lentiviral packaging system background

Review the principles of efficient lentiviral vector packaging and pseudotyping with VSV-G envelope protein to prepare for downstream transfection and transduction.

▶ 02:28
2
Transfect 293TN cells with packaging plasmids

Co-transfect 293TN cells with lentivector expression construct, packaging plasmids, and envelope plasmid using standard transfection methodology to initiate viral particle production.

▶ 03:31
3
Concentrate viral supernatant from transfected cells

Harvest and concentrate lentiviral particles from cell culture supernatant using centrifugation or precipitation to obtain high-titer viral stock.

▶ 05:47
4
Transduce HT1080 cells and determine viral titer

Use serial dilutions of concentrated virus to transduce HT1080 control cells, then quantify transduction efficiency to establish functional viral titer.

▶ 07:36
5
Evaluate successful transfection and transduction

Assess expression of reporter constructs or effector molecules in transduced cells to confirm successful viral packaging and target cell transduction.

▶ 09:21
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