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...
Saved in:
| Main Authors: | , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Taylor & Francis Group
2017-11-01
|
| Series: | BioTechniques |
| Subjects: | |
| Online Access: | https://www.future-science.com/doi/10.2144/000114606 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1850153834000678912 |
|---|---|
| author | Kerstin Laib Sampaio Anja Weyell Narmadha Subramanian Zeguang Wu Christian Sinzger |
| author_facet | Kerstin Laib Sampaio Anja Weyell Narmadha Subramanian Zeguang Wu Christian Sinzger |
| author_sort | Kerstin Laib Sampaio |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-01ce051940fe44bb94411db2bf05e563 |
| institution | OA Journals |
| issn | 0736-6205 1940-9818 |
| language | English |
| publishDate | 2017-11-01 |
| publisher | Taylor & Francis Group |
| record_format | Article |
| series | BioTechniques |
| spelling | doaj-art-01ce051940fe44bb94411db2bf05e5632025-08-20T02:25:37ZengTaylor & Francis GroupBioTechniques0736-62051940-98182017-11-0163520521410.2144/000114606A TB40/E-derived human cytomegalovirus genome with an intact US-gene region and a self-excisable BAC cassette for immunological researchKerstin Laib Sampaio0Anja Weyell1Narmadha Subramanian2Zeguang Wu3Christian Sinzger41Institute of Virology, University of Ulm, Ulm, Germany1Institute of Virology, University of Ulm, Ulm, Germany1Institute of Virology, University of Ulm, Ulm, Germany1Institute of Virology, University of Ulm, Ulm, Germany1Institute of Virology, University of Ulm, Ulm, GermanyFor 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.https://www.future-science.com/doi/10.2144/000114606HCMVTB40/Eself-excisable BAC cassetteUS genesMHC-I |
| spellingShingle | Kerstin Laib Sampaio Anja Weyell Narmadha Subramanian Zeguang Wu Christian Sinzger A TB40/E-derived human cytomegalovirus genome with an intact US-gene region and a self-excisable BAC cassette for immunological research BioTechniques HCMV TB40/E self-excisable BAC cassette US genes MHC-I |
| title | A TB40/E-derived human cytomegalovirus genome with an intact US-gene region and a self-excisable BAC cassette for immunological research |
| title_full | A TB40/E-derived human cytomegalovirus genome with an intact US-gene region and a self-excisable BAC cassette for immunological research |
| title_fullStr | A TB40/E-derived human cytomegalovirus genome with an intact US-gene region and a self-excisable BAC cassette for immunological research |
| title_full_unstemmed | A TB40/E-derived human cytomegalovirus genome with an intact US-gene region and a self-excisable BAC cassette for immunological research |
| title_short | A TB40/E-derived human cytomegalovirus genome with an intact US-gene region and a self-excisable BAC cassette for immunological research |
| title_sort | tb40 e derived human cytomegalovirus genome with an intact us gene region and a self excisable bac cassette for immunological research |
| topic | HCMV TB40/E self-excisable BAC cassette US genes MHC-I |
| url | https://www.future-science.com/doi/10.2144/000114606 |
| work_keys_str_mv | AT kerstinlaibsampaio atb40ederivedhumancytomegalovirusgenomewithanintactusgeneregionandaselfexcisablebaccassetteforimmunologicalresearch AT anjaweyell atb40ederivedhumancytomegalovirusgenomewithanintactusgeneregionandaselfexcisablebaccassetteforimmunologicalresearch AT narmadhasubramanian atb40ederivedhumancytomegalovirusgenomewithanintactusgeneregionandaselfexcisablebaccassetteforimmunologicalresearch AT zeguangwu atb40ederivedhumancytomegalovirusgenomewithanintactusgeneregionandaselfexcisablebaccassetteforimmunologicalresearch AT christiansinzger atb40ederivedhumancytomegalovirusgenomewithanintactusgeneregionandaselfexcisablebaccassetteforimmunologicalresearch AT kerstinlaibsampaio tb40ederivedhumancytomegalovirusgenomewithanintactusgeneregionandaselfexcisablebaccassetteforimmunologicalresearch AT anjaweyell tb40ederivedhumancytomegalovirusgenomewithanintactusgeneregionandaselfexcisablebaccassetteforimmunologicalresearch AT narmadhasubramanian tb40ederivedhumancytomegalovirusgenomewithanintactusgeneregionandaselfexcisablebaccassetteforimmunologicalresearch AT zeguangwu tb40ederivedhumancytomegalovirusgenomewithanintactusgeneregionandaselfexcisablebaccassetteforimmunologicalresearch AT christiansinzger tb40ederivedhumancytomegalovirusgenomewithanintactusgeneregionandaselfexcisablebaccassetteforimmunologicalresearch |