Journal: Scientific Reports

Article Title: Rosa26-LSL-dCas9-VPR: a versatile mouse model for tissue specific and simultaneous activation of multiple genes for drug discovery

doi: 10.1038/s41598-022-23127-7

Figure Lengend Snippet: Generation and characterization of Cre-dependent dCas9-VPR-expressing mice. ( A ) Scheme of the Cre-dependent dCas9-VPR Rosa26 targeting vector. ( B ) Outline of the in vivo experiment. Rosa26-LSL-dCas9-VPR mice were iv injected with AAV8 containing LP1-Cre gene (AAV8- Cre ) or 6 different gRNAs against Pcsk9 (AAV8- gPcsk9 ), named gPcsk9-1 to 6 , at the amount of 1 × 10 11 VG/mouse. 21 days later mice were sacrificed, and tissues were collected for analysis. ( C ) PCR representation showing LSL cassette recombination in liver tissues isolated from Rosa26-LSL-dCas9-VPR mice transduced with AAV8- Cre or AAV8- gPCSK9 alone. A representative agarose gel electrophoresis image is shown. Lane 1–3 contains amplicons obtained from tissue samples of three different mice treated with AAV8- gPcsk9 and lanes 4–6 from tissue samples of three different mice treated with AAV8- Cre . The expected size of PCR products, marker (M) and NTC (H2O) are indicated. ( D ) dCas9-VPR RNA expression in tissues dissected from AAV8- Cre or AAV8- gPCSK9 injected Rosa26-LSL-dCas9-VPR mice. dCas9-VPR expression can only be seen in AAV8- Cre treated Rosa26-LSL-dCas9-VPR mice liver samples. Mean values are shown as a relative quantification, with corresponding expression level of AAV- Cre treated control group as a reference (n = 10).

Article Snippet: AAV8 particles were resuspended in 0.9% NaCl saline (04671613, Deltamedica).

Techniques: Expressing, Plasmid Preparation, In Vivo, Injection, Isolation, Transduction, Agarose Gel Electrophoresis, Marker, RNA Expression, Quantitative Proteomics, Control

Journal: Scientific Reports

Article Title: Rosa26-LSL-dCas9-VPR: a versatile mouse model for tissue specific and simultaneous activation of multiple genes for drug discovery

doi: 10.1038/s41598-022-23127-7

Figure Lengend Snippet: Multiple gRNAs facilitate the transcription of the endogenous mouse Serpina1(a-e) genes in vivo. ( A ) Relative position of the 6 individual gRNAs (black bars) per Serpina1 (a-e) paralogue, aiming to upregulate transcription, are shown. The transcription start site (TSS) in the promoter region is shown as a black arrow. The numbers indicate the base pair distance to the TSS. The position where individual gRNAs bind to the genome is displayed relative to the TSS of the respective Serpina1 transcript (Serpina1a-203 (ENSMUST00000124717.2), Serpina1b-201 (ENSMUST00000095450.11), Serpina1c-201 (ENSMUST00000074051.6), Serpina1e-202 (ENSMUST00000122229.8), Serpina1d-201 (ENSMUST00000078869.6)). ( B ) Outline of the in vivo experiment. Rosa26-LSL-dCas9-VPR mice were iv injected with AAV8 containing LP1-Cre (AAV8- Cre ) alone or in parallel with a set of 5 different AAV8s, carrying gRNAs against 5 Serpina1 paralogues, at the amount of 1 × 10 11 VG/mouse. 21 days later mice were sacrificed, and tissues were collected for analysis. ( C ,D) Serpina1(a-e) mRNA and protein expression in liver tissue dissected from Rosa26-LSL-dCas9-VPR mice, treated with only AAV8- Cre or with a set of six different viruses, AAV8- Cre , AAV8- gSerpina1a , AAV8- gSerpina1b , AAV8- gSerpina1c , AAV8- gSerpina1d and AAV8- gSerpina1e . Mean values are shown as a relative quantification, with corresponding expression level of AAV- Cre treated control group as a reference (n = 9–10). ( E ) AAT protein levels in mouse plasma from Rosa26-LSL-dCas9-VPR mice. Means and standard deviations are shown, n = 10.

Article Snippet: AAV8 particles were resuspended in 0.9% NaCl saline (04671613, Deltamedica).

Techniques: In Vivo, Injection, Expressing, Quantitative Proteomics, Control, Clinical Proteomics

Journal: Scientific Reports

Article Title: Rosa26-LSL-dCas9-VPR: a versatile mouse model for tissue specific and simultaneous activation of multiple genes for drug discovery

doi: 10.1038/s41598-022-23127-7

Figure Lengend Snippet: Parallel Ldlr and Pcsk9 overexpression in vivo using AAV8-transduced CRISPRa mice. ( A ) Relative localization of the 6 individual gRNAs (black bars) targeting mouse Pcsk9 and Ldlr genes are depicted. The transcription start site (TSS) in the promoter regions are shown as black arrows. The numbers indicate the distance in base pair relative to the TSS. The position where individual gRNAs bind to the genome is displayed relative to TSS of the respective Ldlr and Pcsk9 transcripts (Ldlr-201 (ENSMUST00000034713.9), Pcsk9-201 (ENSMUST00000049507.6)). ( B ) Outline of the in vivo experiment. Rosa26-LSL-dCas9-VPR mice were iv injected with AAV8 containing LP1-Cre (AAV8- Cre ) alone or in combination with AAV8- gLdlr , AAV8- gPcsk9 or a mixture of both, at the amount of 1 × 10 11 VG/mouse. 21 days later mice were sacrificed, and tissues were collected for analysis. ( C , D ) Ldlr and Pcsk9 mRNA expression in liver tissue isolated from Rosa26-LSL-dCas9-VPR mice treated with AAV8- Cre alone, or in combination with AAV8- gLdlr , AAV8- gPcsk9 or a mixture of both. Mean values are shown as a relative quantification, with corresponding expression level of AAV- Cre treated control group as a reference (n = 9–10). ( E ) Ldlr protein expression in liver tissue isolated from Rosa26-LSL-dCas9-VPR mice treated with AAV8- Cre alone, or in combination with AAV8- gLdlr , AAV8- gPcsk9 or a mixture of both. Mean values are shown as a relative quantification, with corresponding expression level of AAV- Cre treated control group as a reference (n = 9–10). ( F ) Pcsk9 protein expression in liver tissue isolated from Rosa26-LSL-dCas9-VPR mice were treated with AAV8- Cre alone, or in combination with AAV8- gLdlr , AAV8- gPcsk9 or a mixture of both. Mean values are shown as a relative quantification, with corresponding expression level of AAV- Cre treated control group as a reference (n = 10). ( G ) PCSK9 protein levels detected in mouse plasma from Rosa26-LSL-dCas9-VPR mice. Means and standard deviations are shown, n = 10. ( H ) Representative images of anti-LDL receptor stained liver sections showing differential LDLR expression in Rosa26-LSL-dCas9-VPR mice injected with AAV- Cre alone, together with AAV- gLdlr or AAV8- gPcsk9 , or simultaneous treatment. Upper panel scale bar, 500 µm. Lower panel, magnified, scale bar, 100 µm.

Article Snippet: AAV8 particles were resuspended in 0.9% NaCl saline (04671613, Deltamedica).

Techniques: Over Expression, In Vivo, Injection, Expressing, Isolation, Quantitative Proteomics, Control, Clinical Proteomics, Staining

Journal: Scientific Reports

Article Title: Rosa26-LSL-dCas9-VPR: a versatile mouse model for tissue specific and simultaneous activation of multiple genes for drug discovery

doi: 10.1038/s41598-022-23127-7

Figure Lengend Snippet: Plasma LDL, HDL and cholesterol concentrations in Rosa26-LSL-dCas9-VPR mice after AAV8- gLdlr/gPcsk9 mediated upregulation of hepatic LDLR and/or PCSK9. ( A , B ) Concentrations of LDL and HDL in plasma from Rosa26-LSL-dCas9-VPR mice. Mean values of concentrations and standard deviations are shown, n = 10. ( C ) Total cholesterol concentrations in plasma from Rosa26-LSL-dCas9-VPR mice. Means and standard deviations are shown, n = 10.

Article Snippet: AAV8 particles were resuspended in 0.9% NaCl saline (04671613, Deltamedica).

Techniques: Clinical Proteomics