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. Author manuscript; available in PMC: 2009 Aug 1.

Published in final edited form as: Virology. 2008 Jun 5;377(2):379–390. doi: 10.1016/j.virol.2008.04.034

A) The genomic structure of the regulatory immediate early genes IE1 and IE2 of HCMV. IE1 is composed of 4 exons (exon 1, 2, 3 and 4) indicated by solid dark lines and three introns as indicated by intervening thin lines; IE2 is also composed of 4 exons (exon1, 2, 3 and 5) as indicated by solid dark lines and three introns as indicated by intervening thin lines. B) Construction of IEfusion gene. Primers a, b, c, d, e are described in M&M. IE1/e4 was amplified from the IE1 gene using primers a and b, and was further extended using primer a and c to introduce an internal Apa I site, and external Pme I and Asc I sites. IE2/e5 was amplified from the IE2 gene using primers d and e. It was digested at the newly created Apa I and synthetic Asc I site. IE1/e4 and IE2/e5 were joined together by ligation preserving the reading frame. C) Schematic map of IEfusion-pZWIIA and pp65-IEfusion-pZWIIA MVA transfer plasmids. pZWIIA, an ampicillin resistant plasmid (amp shown in light grey) inserts DNA sequence within the boundaries of MVA Deletion II via Flanking regions 1 and 2 shown in red (FL1, FL2). pZWIIA has two vaccinia synthetic E/L promoters (green) of slightly different sequence, arranged head to head to each drive expression of separate genes. IEfusion gene shown in yellow expression is driven by Psyn I promoter(Chakrabarti, Sisler, & Moss, 1997) and pp65 gene shown in yellow expression is driven by Psyn II promoter(Wyatt et al., 2004). gus bacterial marker gene shown in blue used for identifying recombinant MVA is flanked by two direct repeat (DR) sequences shown in dark grey to facilitate gus gene removal by intragenomic recombination from IEfusion-MVA or pp65-IEfusion-MVA. In neither transfer plasmid was pp65 fused to the IEfusion gene. D) Generation of IEfusion-MVA and pp65-IEfusion-MVA. IEfusion-pZWIIA or pp65-IEfusion-pZWIIA was transfected into wtMVA infected CEF cells to generate IEfusion-MVA or pp65-IEfusion-MVA via homologous recombination at Deletion II whose flanking region is contained in the plasmid that is homologous to wtMVA.

A) The genomic structure of the regulatory immediate early genes IE1 and IE2 of HCMV. IE1 is composed of 4 exons (exon 1, 2, 3 and 4) indicated by solid dark lines and three introns as indicated by intervening thin lines; IE2 is also composed of 4 exons (exon1, 2, 3 and 5) as indicated by solid dark lines and three introns as indicated by intervening thin lines. B) Construction of IEfusion gene. Primers a, b, c, d, e are described in M&M. IE1/e4 was amplified from the IE1 gene using primers a and b, and was further extended using primer a and c to introduce an internal Apa I site, and external Pme I and Asc I sites. IE2/e5 was amplified from the IE2 gene using primers d and e. It was digested at the newly created Apa I and synthetic Asc I site. IE1/e4 and IE2/e5 were joined together by ligation preserving the reading frame. C) Schematic map of IEfusion-pZWIIA and pp65-IEfusion-pZWIIA MVA transfer plasmids. pZWIIA, an ampicillin resistant plasmid (amp shown in light grey) inserts DNA sequence within the boundaries of MVA Deletion II via Flanking regions 1 and 2 shown in red (FL1, FL2). pZWIIA has two vaccinia synthetic E/L promoters (green) of slightly different sequence, arranged head to head to each drive expression of separate genes. IEfusion gene shown in yellow expression is driven by Psyn I promoter(Chakrabarti, Sisler, & Moss, 1997) and pp65 gene shown in yellow expression is driven by Psyn II promoter(Wyatt et al., 2004). gus bacterial marker gene shown in blue used for identifying recombinant MVA is flanked by two direct repeat (DR) sequences shown in dark grey to facilitate gus gene removal by intragenomic recombination from IEfusion-MVA or pp65-IEfusion-MVA. In neither transfer plasmid was pp65 fused to the IEfusion gene. D) Generation of IEfusion-MVA and pp65-IEfusion-MVA. IEfusion-pZWIIA or pp65-IEfusion-pZWIIA was transfected into wtMVA infected CEF cells to generate IEfusion-MVA or pp65-IEfusion-MVA via homologous recombination at Deletion II whose flanking region is contained in the plasmid that is homologous to wtMVA.