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: LSL cassette recombination PCR was performed on liver cDNA using Quick-Load® Taq Master Mix (M0271S, NEB) with primers p1, p3, p2 (Supplementary Table ).
Techniques: Expressing, Plasmid Preparation, In Vivo, Injection, Isolation, Transduction, Agarose Gel Electrophoresis, Marker, RNA Expression, Quantitative Proteomics, Control
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