Journal: Viruses

Article Title: A Lac Repressor-Inducible Baculovirus Expression Vector for Controlling Adeno-Associated Virus Capsid Ratios

doi: 10.3390/v16010051

Figure Lengend Snippet: An Original and Commonly Used rBEV (Bacmid) RepCap Dual Cassette Design for Producing rAAV Sf9 Cells. The overlapping Rep78/Rep52 and Cap-related VP1/VP2/VP3/AAP genes are shown in context with the bipartite baculovirus very late polh / p10 promoter driving expression of Rep and Cap. This 4389 bp dual cassette is cloned into a single location in the BEV. Non-canonical translation start-site sequences are indicated. Rep genes Rep78 (1866 bp) and Rep52 (1194 bp) have common ORFs and 3′ regions. Cap genes VP1 (2111 bp), VP2 (1800 bp), and VP3 (1605 bp) have common ORF 3′ regions. The AAP gene ORF is out of frame from Cap genes, begins 526 bp from the VP1 translational initiation site, and is 594 bp. Only the VP1 and VP2 ORFs overlap the AAP ORF.

Article Snippet: A synthetically made (GenScript) AvrII- polh promoter-LacR ORF-AvrII cassette was ligated using T4 ligase.

Techniques: Expressing, Clone Assay

Journal: Viruses

Article Title: A Lac Repressor-Inducible Baculovirus Expression Vector for Controlling Adeno-Associated Virus Capsid Ratios

doi: 10.3390/v16010051

Figure Lengend Snippet: Comparison of the Original bMON14272 Bacmid and VoyBac1.1 Bacmid Backbones. The 142,272 bp pMON14272 bacmid ( A ) and the 140,505 bp Voyager VoyBac1.1 bacmid ( B ) are schematically illustrated. Both bacmids have the same min-AttnTn7 transposition elements and a 6619-bp miniF replicon. The location of Tn7 transposition insertion is indicated by Tn7. The v-cath and ChiA deletion regions in VoyBac1.1 are indicated as VC. Baculovirus genes surrounding Tn7, FseI, AvrII, and I-CeuI sites and their directions of transcription are also illustrated.

Article Snippet: A synthetically made (GenScript) AvrII- polh promoter-LacR ORF-AvrII cassette was ligated using T4 ligase.

Techniques: Comparison

Journal: Viruses

Article Title: A Lac Repressor-Inducible Baculovirus Expression Vector for Controlling Adeno-Associated Virus Capsid Ratios

doi: 10.3390/v16010051

Figure Lengend Snippet: Testing LacR Regulation of LacOVP1 Expression in the Context of Baculovirus Infection. Bacmid VB-LacR-LacOVP1 is schematically illustrated ( A ). The location and transcription directions of baculovirus genes gp64 , gta , and Lef1 are shown. This bacmid included a LacO-p10-LacO promoter VP1 cassette ( B ) in the VC locus and a polh promoter LacR cassette in the egt locus ( C ). Sf9 cells were infected at 10 MOI in the presence of different concentrations of IPTG. At 72 hpi, infected Sf9 cells were collected, fractionated by SDS-PAGE, and then Western blot probed with an anti-capsid monoclonal antibody ( D ) and an anti-lac repressor antibody ( E ). Relative abundances of ECL-detected proteins were quantified using ImageJ software (version 1.53) and plotted ( F ).

Article Snippet: A synthetically made (GenScript) AvrII- polh promoter-LacR ORF-AvrII cassette was ligated using T4 ligase.

Techniques: Expressing, Infection, SDS Page, Western Blot, Software

Journal: Viruses

Article Title: A Lac Repressor-Inducible Baculovirus Expression Vector for Controlling Adeno-Associated Virus Capsid Ratios

doi: 10.3390/v16010051

Figure Lengend Snippet: Testing bacmid VB-LacR-Rep-VP3 and Validating a Hybrid Early/Late/Very Late Promoter for LacR Expression. The bacmid VB-LacR-Rep-VP3 is schematically illustrated ( A ). This bacmid was based on VoyBac1.1 and had a LacR-Rep-VP3 multi-cassette inserted into the Tn7 locus. The Rep and VP3 were in opposing orientations and had polh and p10 promoters, respectively ( B ). The LacR ORF was downstream of Rep and in the same orientation. The original promoter design for LacR in this multi-cassette was a hybrid Opgp64 - polh promoter ( C ). This promoter had two late ATAAG transcriptional initiation sites: an early CAGT transcriptional initiation site in the Opgp64 promoter and a very late ATAAG transcriptional initiation site in the polh promoter. A minicistron was present in the polh promoter upstream of the polh ATAAG transcriptional initiation site ( D ). A mutagenic PCR primer was designed to remove two ATG translational initiation sites in the minicistron ( B ). A mutagenic PCR primer was used to remove the minicistron translational initiation sites from the polh promoter, resulting in an Opgp64polh mt LacR cassette ( E ). Two versions of VB-LacR-Rep-VP3 were made: Opgp64polh wt LacR-Rep-VP3 (original version) and Opgp64polh mt LacR-Rep-VP3 (minicistron mutated version). Sf9 cells were infected at 10 MOI with the two versions of bacmid VB-LacR-Rep-VP3, and insect cells were collected at different hours post-infection. Cell proteins were fractionated by SDS-PAGE, and then Western blots were probed with anti-Cap or anti-LacR antibodies. The relative abundances of Western-detected VP3 ( F ) and LacR ( G ) are plotted on the graphs.

Article Snippet: A synthetically made (GenScript) AvrII- polh promoter-LacR ORF-AvrII cassette was ligated using T4 ligase.

Techniques: Expressing, Infection, SDS Page, Western Blot