Journal: mSphere

Article Title: Control of Human Anelloviruses by Cytosine to Uracil Genome Editing

doi: 10.1128/msphere.00506-22

Figure Lengend Snippet: Presence of uracil in packaged anellovirus genomes. Ethidium bromide-stained agarose gel with PCR products generated by 3D-PCR targeting TTMV-AMS-S1-44. The primers for product 1 ( Fig. 1D to ) were used on nucleic acids from subject 1 collected at start of study (time point 1, TP1). Samples were treated with (+) or without (−) USER enzyme (Uracil-Specific Excision Reagent), which can catalyze the excision of the uracil base and break the phosphodiester backbone of the products. Synthetic oligonucleotides were used as controls containing the DNA sequence of product 1 with 0 mutations (control unedited reference), 6 C-to-T mutations (50% editing; control edited C > T) or 6 C-to-U mutations (50% editing) (control edited C > U). In addition, a 1:1 mixture of products containing the control unedited reference and control edited C-to-U mutations was included (control mix reference + C > U) to mimic the mixture present in the serum sample. Products visible under 100 base pairs (bp) did not have the expected length and were considered primer dimers. Unprocessed agarose gel is shown in Fig. S2 . M, 100-bp ladder.

Article Snippet: Several synthetic DNA sequences (Biolegio, Nijmegen, Netherlands) were used as controls at an input of 10 5 copies per reaction.

Techniques: Staining, Agarose Gel Electrophoresis, Generated, Sequencing, Control

Journal: mSphere

Article Title: Control of Human Anelloviruses by Cytosine to Uracil Genome Editing

doi: 10.1128/msphere.00506-22

Figure Lengend Snippet: Editing hot- and coldspots. (A) Frequency of editing in TTMV-AMS-S1-41 per possible editing position, scored by the number of time points which show editing ( y axis) on the coding strand of the genome ( x axis). Schematic overview of open reading frame (ORF) locations; GC-rich region in TTMV-AMS-S1-41 is shown to clarify ORF locations. Asterisks (*) indicate editing coldspots. (B) Negative-stranded DNA C nucleotide context for 3 lineage-specific editing coldspots. (C) Editing frequencies in ORFs and GC-rich regions calculated for all PTE lineages. Differences in mean mutation frequency were tested using a Kruskal-Wallis test followed by Dunn’s multiple-comparison test. (D) Negative-stranded DNA C nucleotide context in the GC-rich region of all PTE lineages per genus: TTV, TTMV, and TTMDV. (E) Fraction of editing-based substitutions that are synonymous (Syn) or nonsynonymous (NS). Each dot represents a PTE lineage at an EAT. A paired t test was used to compare mean frequency between synonymous and nonsynonymous substitutions. ****, P ≤ 0.0001.

Article Snippet: Several synthetic DNA sequences (Biolegio, Nijmegen, Netherlands) were used as controls at an input of 10 5 copies per reaction.

Techniques: Mutagenesis, Comparison

Journal: Cell & Bioscience

Article Title: A minimal bile salt excretory pump promoter allows bile acid-driven physiological regulation of transgene expression from a gene therapy vector

doi: 10.1186/s13578-022-00803-9

Figure Lengend Snippet: AAV vectors expressing LucPEST downstream of BSEP minimal promoters. Schematic representation of AAV vectors containing a BSEP minimal promoter from human (ihPr) ( a ) and mouse (imPr) ( b ) origin. ITR: inverted terminal repeats; LucPEST: destabilized firefly luciferase sequence; pA: synthetic polyadenylation signal

Article Snippet: Synthetic DNA sequences of minimal BSEP promoters from mouse (imPr) and human (ihPr) origin (Additional file : Fig. S1) were based on human and mouse full-length BSEP promoters, respectively (NCBI Reference Sequences: AF190696.1 and AF190697.1, respectively) and obtained from GenScript (Nanjing, China) cloned into pUC57 flanked by Mlu I and Nhe I sites (pUC57-imPr & pUC57-ihPr).

Techniques: Expressing, Luciferase, Sequencing

Journal: Cell & Bioscience

Article Title: A minimal bile salt excretory pump promoter allows bile acid-driven physiological regulation of transgene expression from a gene therapy vector

doi: 10.1186/s13578-022-00803-9

Figure Lengend Snippet: Bile acid induction of luciferase expression in human hepatic cell lines. HepG2 ( a ) and Huh-7 ( b ) cells were transfected with plasmids expressing LucPEST downstream of the indicated promoters with or without co-transfection of the human FXRα2 isoform and with or without incubation with chenodeoxycholic acid (CDCA). All samples were tested in triplicates and data are presented as mean ± SEM of relative (rel.) units (Luciferase units sec −1 /Renilla units sec −1 ). The fold induction of each condition relative to cells transfected only with the various luciferase plasmids (pLuc) is indicated above each bar, as well as the statistical analysis comparing these two conditions. Other comparisons are shown by horizontal bars. *p < 0.05; **p < 0.01; ***p < 0.001; ns: not significant. imPr, pAAV-imPr-LucPEST; ihPr, pAAV-ihPr-LucPEST; full-length mPr, pAAV-mBSEPpr-LucPEST; full-length hPr, p-2563/ + 4-Luc; A1AT, pAAV-A1AT-LucPEST

Article Snippet: Synthetic DNA sequences of minimal BSEP promoters from mouse (imPr) and human (ihPr) origin (Additional file : Fig. S1) were based on human and mouse full-length BSEP promoters, respectively (NCBI Reference Sequences: AF190696.1 and AF190697.1, respectively) and obtained from GenScript (Nanjing, China) cloned into pUC57 flanked by Mlu I and Nhe I sites (pUC57-imPr & pUC57-ihPr).

Techniques: Luciferase, Expressing, Transfection, Cotransfection, Incubation

Journal: Cell & Bioscience

Article Title: A minimal bile salt excretory pump promoter allows bile acid-driven physiological regulation of transgene expression from a gene therapy vector

doi: 10.1186/s13578-022-00803-9

Figure Lengend Snippet: Bile acid induction of minimal BSEP promoters in wild-type (WT) mice. C57BL/6 male and female mice were administered 3 × 10 12 VG/kg of AAV8-imPr-LucPEST ( a ), AAV8-A1AT-LucPEST ( b ), or AAV8-ihPr-LucPEST ( d ) and received either normal diet (red open squares) or a diet supplemented with 0.2% cholic acid (CA) (blue closed circles) alternating with normal diet (blue open circles) as indicated in the figure. Luciferase expression was measured in live mice at the indicated times (n = 3 for each gender except in mice receiving AAV-ihPr-LucPEST where n = 2, only tested in females). c Representative bioluminiscence images of C57BL/6 male mice administered with AAV8-imPr-LucPEST and AAV8-A1AT-LucPEST (n = 3) that received alternating CA/normal diets. Images were taken at week 13 before the last CA cycle and at week 16 after 3 weeks of CA diet in the third cycle. Data are shown as mean + SEM of photon (Ph) units per second normalized with background signal. Statistical comparisons were calculated comparing the average luciferase expression during each CA cycle and its precedent normal diet cycle using a paired T test. *p < 0.05; **p < 0.01. Fold induction in each CA cycle was calculated by dividing the maximum expression under CA induction by basal expression prior to CA administration. imPr, AAV8-imPr-LucPEST; ihPr, AAV8-ihPr-LucPEST; A1AT, AAV-A1AT-LucPEST

Article Snippet: Synthetic DNA sequences of minimal BSEP promoters from mouse (imPr) and human (ihPr) origin (Additional file : Fig. S1) were based on human and mouse full-length BSEP promoters, respectively (NCBI Reference Sequences: AF190696.1 and AF190697.1, respectively) and obtained from GenScript (Nanjing, China) cloned into pUC57 flanked by Mlu I and Nhe I sites (pUC57-imPr & pUC57-ihPr).

Techniques: Luciferase, Expressing

Journal: Cell & Bioscience

Article Title: A minimal bile salt excretory pump promoter allows bile acid-driven physiological regulation of transgene expression from a gene therapy vector

doi: 10.1186/s13578-022-00803-9

Figure Lengend Snippet: Luciferase expression in Abcb4 −/− mice. Abcb4 −/− mice exhibit high levels of bile acids in serum, making them a good model to test the physiological inducibility of minimal BSEP promoters. FVB Abcb4 −/− and WT male and female mice were administered 3 × 10 12 VG/kg of AAV8-imPr-LucPEST ( a ), AAV8-A1AT-LucPEST ( b ) or AAV8-ihPr-LucPEST ( c ). Luciferase was measured in live mice at the indicated times (n = 3 for each gender and vector, except in knock-out (KO) males inoculated with AAV-imPr-LucPEST where n = 4). The statistical comparison between Abcb4 −/− and WT mice was performed by using an unpaired T test at each time point. *p < 0.05; **p < 0.01; ***p < 0.001. Data are shown as mean ± SEM of photon (Ph) units per second normalized with background signal. imPr, AAV8-imPr-LucPEST; ihPr, AAV8-ihPr-LucPEST; A1AT, AAV8-A1AT-LucPEST

Article Snippet: Synthetic DNA sequences of minimal BSEP promoters from mouse (imPr) and human (ihPr) origin (Additional file : Fig. S1) were based on human and mouse full-length BSEP promoters, respectively (NCBI Reference Sequences: AF190696.1 and AF190697.1, respectively) and obtained from GenScript (Nanjing, China) cloned into pUC57 flanked by Mlu I and Nhe I sites (pUC57-imPr & pUC57-ihPr).

Techniques: Luciferase, Expressing, Plasmid Preparation, Knock-Out, Comparison

Journal: Cell & Bioscience

Article Title: A minimal bile salt excretory pump promoter allows bile acid-driven physiological regulation of transgene expression from a gene therapy vector

doi: 10.1186/s13578-022-00803-9

Figure Lengend Snippet: BA induction of an optimized minimal BSEP promoter in wild type mice. C57BL/6 male and female mice were administered 3 × 10 12 VG/kg of AAV8-imPr-3xIR-LucPEST or AAV8-imPr-LucPEST and received either a diet supplemented with 0.2% CA (closed symbols) alternating with normal diet (open symbols) ( a ) or a continuous normal diet ( b ). Luciferase was measured in live mice at the indicated times (n = 4). Data are shown as mean + SEM of photon (Ph) units per second normalized to background signal. The statistical comparisons and fold induction were calculated as described in Fig. a (indicated in the upper part of the graph for imPr-3xIR and in the lower part for imPr). imPr, AAV8-imPr-LucPEST; imPr-3xIR, AAV8-imPr-3xIR-LucPEST

Article Snippet: Synthetic DNA sequences of minimal BSEP promoters from mouse (imPr) and human (ihPr) origin (Additional file : Fig. S1) were based on human and mouse full-length BSEP promoters, respectively (NCBI Reference Sequences: AF190696.1 and AF190697.1, respectively) and obtained from GenScript (Nanjing, China) cloned into pUC57 flanked by Mlu I and Nhe I sites (pUC57-imPr & pUC57-ihPr).

Techniques: Luciferase