Journal: Nature Communications

Article Title: Viral gene drive in herpesviruses

doi: 10.1038/s41467-020-18678-0

Figure Lengend Snippet: Fibroblasts were coinfected with Towne-eGFP (WT) and GD-mCherry, and supernatant was used to infect fresh cells. a Representative examples of fluorescent viral plaques spreading on fibroblasts, expressing either eGFP only (left), mCherry only (middle), or both (right). Scale bar: 100 μm. b PCR for mCherry (upper band) and eGFP (lower band) on 48 recombinant viral genomes from two independent experiments. c Same as ( b ), after coinfection with GD-mCherry and a mutated Towne-eGFP (two biological replicates, 30 genomes). d PCR of homology arms and Sanger sequencing of 17 eGFP-mCherry expressing viral clones. Blue dots: single nucleotide polymorphisms (SNPs) from Towne strain; Red dots: TB40/E strain. e Fraction of SNPs of Towne or TB40/E origin alongside the hCMV genome. Each dot represents an individual SNP. Data combine two biological replicates after Oxford Nanopore sequencing. Coverage gives the number of reads, allowing multiple mapping. f Reconstruction of recombination history of individual hCMV genomes from long (>200 kb) Nanopore reads. One genome corresponds to one sequencing read and colors indicate the strain of origin. Gaps represent regions deleted compared to the reference genome, dashed lines indicate uncovered region. UL/US: unique long/short genome segments. GeneRuler 1 kb DNA ladder (ThermoFisher) was used in agarose gels as a size marker. The ladder is shown in the gel images, with the stronger band corresponding to 500 bp. Source data are provided as a Source Data file.

Article Snippet: All modifications were carried out by Gibson cloning (NEB, Ipswich, MA, USA), using PCR products from other plasmids or synthesized DNA fragments (GeneArt™ String™ fragments, ThermoFisher, USA).

Techniques: Expressing, Recombinant, Sequencing, Clone Assay, Nanopore Sequencing, Marker