Home › Immunology › Generating Recombinant Avian Herpesvirus Vectors with CRISPR/Cas9 Gene Editing
Immunology JoVE (Open Access) Citable · DOI

Generating Recombinant Avian Herpesvirus Vectors with CRISPR/Cas9 Gene Editing

DOI: 10.3791/58193-v
What you'll learn
  • ✓ Design CRISPR/Cas9-mediated knock-in constructs
  • ✓ Perform transfection and infection for recombinant HVT
  • ✓ Purify recombinant HVT from infected cells
  • ✓ Excise fluorescent reporter using Cre-lox system
Protocol

Biopharma Insights Herpesvirus of turkeys (HVT) is widely used as a vector platform for the generation of recombinant vaccines against a number of avian diseases. This article describes a simple and rapid approach for the generation of recombinant HVT-vectored vaccines using an integrated NHEJ-CRISPR/Cas9 and Cre-Lox system.

Difficulty
advanced
Total time
~2-3 weeks for complete protocol
Model organism
Chicken embryo fibroblasts (CEF)
Biosafety
BSL-2

Steps

1
Transfect cells with CRISPR/Cas9 and infect

Introduce CRISPR/Cas9 components into cells and subsequently infect with HVT to induce gene editing.

▶ 00:53
2
Harvest and purify recombinant HVT

Collect infected cells and purify the recombinant virus from the culture.

▶ 02:14
3
Excise fluorescent reporter using Cre-lox

Treat the recombinant virus with Cre recombinase to remove the fluorescent marker gene.

▶ 05:38
4
Plaque purify recombinant virus

Perform plaque purification to isolate clonal recombinant virus populations.

▶ 06:23
5
Verify recombinant HVT genome

Confirm the correct insertion and structure of the recombinant virus through molecular analysis.

▶ 07:31
6
Assess stability of recombinant virus

Evaluate the genetic stability of the recombinant virus over multiple passages.

▶ 09:31
7
Analyze results of generated vectors

Examine the outcomes of the recombinant avian herpesvirus vector generation.

▶ 10:36
8
Conclude protocol and applications

Summarize the integrated CRISPR/Cas9 and Cre-lox approach for generating recombinant HVT vaccines.

▶ 11:38
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