| Autor: |
Weiler, Nina, Sampaio, Kerstin Laib, Scherer, Myriam, Sinzger, Christian |
| Zdroj: |
Biotechniques; November 2023, Vol. 75 Issue: 5 p183-194, 12p |
| Abstrakt: |
Working with recent isolates of human cytomegalovirus (HCMV) is complicated by their strictly cell-associated growth with lack of infectivity in the supernatant. Adaptation to cell-free growth is associated with disruption of the viral UL128gene locus. The authors transduced fibroblasts with a lentiviral vector encoding UL128-specific-shRNA to allow the release of cell-free infectivity without genetic alteration. Transduced cells were cocultured with fibroblasts containing cell-associated isolates, and knockdown of the UL128 protein was validated by immunoblotting. Cell-free infectivity increased 1000-fold in isolate cocultures with UL128-shRNA compared with controls, and virions could be purified by density gradients. Transduced fibroblasts also allowed direct isolation of HCMV from a clinical specimen and cell-free transfer to other cell types. In conclusion, UL128-shRNA-transduced fibroblasts allow applications previously unsuitable for recent isolates. |
| Databáze: |
Supplemental Index |
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