Figure 3 | Scientific Reports

Figure 3

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

Figure 3The alternative text for this image may have been generated using AI.

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 × 1011 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.

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