Exploiting non-permissive CHO cells as a rapid and efficient method for recombinant HSV-1 isolation.
BHK-21
CHO cells
Fluorescent reporter
Non-permissive
Oncolytic HSV-1
Purification plaque
Journal
AMB Express
ISSN: 2191-0855
Titre abrégé: AMB Express
Pays: Germany
ID NLM: 101561785
Informations de publication
Date de publication:
09 May 2024
09 May 2024
Historique:
received:
24
05
2023
accepted:
17
04
2024
medline:
9
5
2024
pubmed:
9
5
2024
entrez:
9
5
2024
Statut:
epublish
Résumé
Using herpes simplex virus type 1 (HSV-1) as a therapeutic tool has recently emerged as a promising strategy for enhancing the treatment of various cancers, particularly those associated with the nervous system, which is the virus's natural site of infection. These viruses are specifically engineered to infect and eradicate tumor cells while leaving healthy cells unharmed. To introduce targeted mutations in specific viral genes, gene-modification techniques such as shuttle vector homologous recombination are commonly employed. Plaque purification is then utilized to select and purify the recombinant virus from the parental viruses. However, plaque purification becomes problematic when the insertion of the desired gene at the target site hampers progeny virus replication, resulting in a lower titer of cell-released virus than the parental virus. This necessitates a laborious initial screening process using approximately 10-15 tissue culture dishes (10 cm), making plaque purification time-consuming and demanding. Although the recently developed CRISPR-Cas9 system significantly enhances the efficiency of homologous integration and editing precision in viral genes, the purification of recombinant variants remains a tedious task. In this study, we propose a rapid and innovative method that employs non-permissive Chinese hamster ovary (CHO) cells, representing a remarkable improvement over the aforementioned arduous process. With this approach, only 1-2 rounds of plaque purification are required. Our proposed protocol demonstrates great potential as a viable alternative to current methods for isolating and purifying recombinant HSV-1 variants expressing fluorescent reporter genes using CHO cells and plaque assays.
Identifiants
pubmed: 38722404
doi: 10.1186/s13568-024-01709-0
pii: 10.1186/s13568-024-01709-0
doi:
Types de publication
Journal Article
Langues
eng
Pagination
53Informations de copyright
© 2024. The Author(s).
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