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

Begin with a mixed population of chick embryo fibroblasts (CEFs) infected with recombinant herpesviruses.

In one population, the viral genome contains both the target gene and the GFP reporter. In the other, it retains only the target gene following site-specific excision of GFP.

Use fluorescence-activated cell sorting to deposit a GFP-negative, infected CEF into the well containing uninfected CEFs.

Incubate to allow viral replication leading to CEF lysis and spread of the virus to neighboring cells.

After incubation, add an enzyme to detach the cells, transfer the suspension to a tube, and centrifuge.

Discard the supernatant and resuspend the pellet in a lysis buffer to release DNA.

Transfer the DNA to a PCR mixture containing gene-specific primers.

Perform PCR to amplify the target gene.

Load the PCR product onto an agarose gel, run electrophoresis, and visualize under UV light.

A correctly sized amplicon corresponding to the target gene confirms its retention in GFP-excised recombinant herpesvirus.

72 hours post infection prepare the cells for sorting as previously described. Sort the single non-fluorescent cells into a 96 well plate preseeded with CEFs. Next, trypsinize the chosen plaques and resuspend the corresponding cells as outlined in the text protocol.

Pass half of the cells into each well of a 96 well plate that has been preseeded with CEFs as the second generation. Centrifuge the remaining cells of each clone at 200 times gravity for five minutes. Discard the supernatant and resuspend the cells with 50 microliters of squishing buffer for DNA extraction.

Then, perform PCR with five prime junction primers using 1 microliter of DNA template as outlined in the text protocol. Based on the PCR results, choose three to five positive clones of recombinant HVT for further passages and verification.