Clonal Isolation and PCR Screening of GFP-Excised Recombinant Herpesvirus Retaining the Target Gene

Résumé

Source: Tang, N., et al. Generating Recombinant Avian Herpesvirus Vectors with CRISPR/Cas9 Gene Editing. J. Vis. Exp. (2019)

This video demonstrates the clonal isolation and PCR-based screening of recombinant herpesvirus following site-specific excision of a GFP marker. Fluorescence-activated cell sorting is used to isolate non-fluorescent infected cells, and junction PCR confirms retention of the target gene in GFP-negative clones.

Protocole

Plaque Purification

1. Fluorescence-activated cell sorting
   1. Seed two 96-well plates with 2 x 10⁴ Chick Embryo Fibroblast (CEF) cells per well the day before sorting.
   2. 72 h postinfection, prepare the infected cells for sorting. Sort the single nonfluorescence cells into 96-well plates seeded with CEFs.
2. Passaging of the recombinant viruses and PCR confirmation
   1. Choose 5 - 10 single nonfluorescence plaques 5 d postsorting, trypsinize them with 50 µL of trypsin-EDTA at 38.5 °C for 3 min, and add 50 µL of culture medium to resuspend the cells.
      NOTE: See Figure 1B for a representative GFP-negative plaque.
   2. Pass half of the cells into each well of a 6-well plate preseeded with CEFs as the second generation.
   3. Centrifuge the remaining cells of each clone at 200 x g for 5 min, discard the supernatant, and resuspend the cells with 50 µL of squishing buffer for DNA extraction.
   4. Perform PCR with 5' junction primers using 1 µL of DNA template. For each sample, prepare the following 20 µL reaction mix on ice: 2x PCR Master Mix (10 µL), 10 µM upstream primer (0.5 µL), 10 µM downstream primer (0.5 µL), DNA template (1 µL), and nuclease-free water (8 µL). The amplification program is: 95 °C for 2 min; 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 40 s for 35 cycles; 72 °C for 7 min. Load 2 µL of the amplification products to one well of 1% agarose gel for gel electrophoresis.
      NOTE: See Figure 1B for a representative result of 5' junction PCR.
   5. Based on the PCR results, choose three to five positive clones of recombinant HVT for further passages and verification.

Résultats

Figure 1: Verification of the recombinant HVT. (A) This panel shows a GFP-positive plaque (HVT-GFP-VP2) visualized under the fluorescence microscope (top panel) and the PCR verification of HVT-GFP-VP2 with primers VP2-F & UL46-R1 for the 3...

Matériaux

Liste des matériaux utilisés dans cet article

Nom	Entreprise	Numéro de catalogue	Commentaires
HVT Fc126 strain	Avian Disease and Oncology Laboratory		wildtype HVT virus
GoTaq Master Mixes	Promega	M7123	junction PCR amplification
Platinum Pfx DNA Polymerase	Thermofisher	11708021	PCR amplification of full insert
Confocal laser scanning microscope	Leica Microsystems	Leica TCS SP5 LASAF	immunoflourescence
FACS cell sorter	BD biosciences	BD FACSAria	single cell sorting