Home › Microbiology › Production, Purification, and Quality Control for Adeno-associated Virus-based Vectors
Microbiology JoVE (Open Access) Citable · DOI

Production, Purification, and Quality Control for Adeno-associated Virus-based Vectors

DOI: 10.3791/58960-v
What you'll learn
  • ✓ Perform tri-transfection of HEK293T cells to produce AAV vectors
  • ✓ Purify AAV vectors using column chromatography techniques
  • ✓ Apply quality control assays to assess vector titer and purity
  • ✓ Generate high-titer (≥1×10¹³ vg/mL) AAV preparations for downstream use
Protocol

Here, we describe an efficient and reproducible strategy to produce, titer, and quality-control batches of adeno-associated virus vectors. It allows the user to obtain a vector preparation with high-titer (≥1 x 1013 vector genomes/mL) and a high purity, ready for in vitro or in vivo use.

Difficulty
advanced
Total time
~5–7 days (transfection, production, purification, and QC analysis)
Model organism
HEK293T
Biosafety
BSL-2

Steps

1
Perform tri-transfection of HEK293T cells

Co-transfect HEK293T cells with three plasmids: AAV rep/cap, transgene, and helper plasmid. This approach generates recombinant AAV particles expressing the desired transgene.

▶ 00:46
2
Purify AAV vectors using column chromatography

Harvest transfected cell lysate and apply purification strategy (e.g., iodixanol density-gradient ultracentrifugation or affinity chromatography) to isolate AAV particles from cellular contaminants and achieve high purity.

▶ 03:59
3
Analyze AAV vectors via quality control assays

Perform titer determination (e.g., qPCR, ELISA) and purity assessment (e.g., SDS-PAGE, transmission electron microscopy) to confirm high-titer and contaminant-free AAV preparation suitable for research use.

▶ 07:28
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