HCMV Strain Production and Infection

The HCMV strain TB40/E WT and the deletion mutant TB40/E ∆US28 were previously described [53 (link)]. HCMV strain Merlin was generated from a bacterial artificial chromosome BAC pAL2157. This is BAC pAL1498 [49 (link)] in which eGFP was linked to the IE2 gene using a P2A linker. SW102 E. coli containing BAC pAL2157 were kindly provided by Dr. Richard J. Stanton. BAC DNA was isolated using NucleoBond Xtra BAC kit (Macherey Nagel, Düren, Germany). Virus production was initiated by electroporation of BAC DNA into HFFF TR cells using the Amaxa Nucleofector and the basic fibroblast nucleofector kit (Lonza, Basel, Switserland). Subsequent virus productions in HFFF TR cells were started with an infection at MOI 0.02 and titres were determined after 3 days using immediate early antigen staining. U251 and GBM48 GBM cells were infected at MOI 3.