A TB40/E-derived human cytomegalovirus genome with an intact US-gene region and a self-excisable BAC cassette for immunological research
For immunological research on the human cytomegalovirus (HCMV), a virus that combines the broad cell tropism of clinical isolates, efficient replication in cell culture, the complete set of MHC-I modulator genes, and suitability for genetic engineering is desired. Here, we aimed to generate a geneti...
| Veröffentlicht in: | BioTechniques. - 1988. - 63(2017), 5 vom: 01. Nov., Seite 205-214 |
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| 1. Verfasser: | |
| Weitere Verfasser: | , , , |
| Format: | Online-Aufsatz |
| Sprache: | English |
| Veröffentlicht: |
2017
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| Zugriff auf das übergeordnete Werk: | BioTechniques |
| Schlagworte: | Journal Article Research Support, Non-U.S. Gov't RNA-Binding Proteins US2 protein, Varicellovirus US6 protein, Human cytomegalovirus Viral Envelope Proteins Viral Proteins enhanced green fluorescent protein Green Fluorescent Proteins 147336-22-9 |
| Zusammenfassung: | For immunological research on the human cytomegalovirus (HCMV), a virus that combines the broad cell tropism of clinical isolates, efficient replication in cell culture, the complete set of MHC-I modulator genes, and suitability for genetic engineering is desired. Here, we aimed to generate a genetically complete derivative of HCMV strain TB40/E as a bacterial artificial chromosome (BAC) with a self-excisable BAC cassette. The BAC cassette was inserted into the US2-US6 gene region (yielding TB40-BACKL7), relocated into the UL73/UL74 region with modifications that favor excision of the BAC cassette during replication in fibroblasts, and finally the US2-US6 region was restored, resulting in BAC clone TB40-BACKL7-SE When this BAC clone was transfected into fibroblasts at efficiencies >0.1%, replicating virus that had lost the BAC cassette appeared within 2 weeks after transfection, grew to high titers, and displayed the broad tropism of the parental virus. The degree of MHC-I down-regulation by this virus was consistent with functional restoration of US2-US6. To enable detection of infected cells by flow cytometry, an enhanced green fluorescent protein (EGFP)-expression cassette was inserted downstream of US34A, yielding the fluorescent virus RV-TB40-BACKL7-SE-EGFP |
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| Beschreibung: | Date Completed 25.07.2018 Date Revised 07.11.2018 published: Electronic Citation Status MEDLINE |
| ISSN: | 1940-9818 |
| DOI: | 10.2144/000114606 |