Journal:

Article Title: Improved Retinal Function in a Mouse Model of Dominant Retinitis Pigmentosa Following AAV-delivered Gene Therapy

doi: 10.1038/mt.2008.301

Figure Lengend Snippet: Schematic representation of the AAVshQ1 suppression construct. shQ1 RNA was expressed from the H1 promoter (H1shQ1). The H1shQ1 cassette was flanked up- and down-stream by spacer DNA fragments. EGFP was coexpressed from the CMV immediate-early promoter (CMVP). The SV40 polyadenylation signal (PolyA) was located at the 3′ end of the EGFP gene. Restriction enzyme sites used for cloning are given. Numbers indicate molecular sizes in base pairs, and arrows indicate the direction of transcription. L-ITR and R-ITR: left and right inverted repeats of AAV.

Article Snippet: The RHO-targeting short hairpin RNA shQ1 (target position nt 650–670; ref. 13 ) was cloned into pAAV-MCS (Stratagene, La Jolla, CA) with the EGFP reporter gene [pEGFP-1, BD Biosciences, Oxford, UK (Entrez Gene accession no. {"type":"entrez-nucleotide","attrs":{"text":"U57608","term_id":"1543069","term_text":"U57608"}} U57608 )] to create pAAVshQ1 ( ). shQ1 targets the RHO nucleotide sequence TCG TGT GGA ATC GAC TAC that differs from the mouse Rho sequence at four positions as underlined, TC A TG C GG G AT T GAC TAC, while encoding the same amino acids.

Techniques: Construct, Clone Assay

Journal:

Article Title: Improved Retinal Function in a Mouse Model of Dominant Retinitis Pigmentosa Following AAV-delivered Gene Therapy

doi: 10.1038/mt.2008.301

Figure Lengend Snippet: Suppression of human and mouse rhodopsin in vivo. Fellow eyes of NHR (RHO+/−Rho−/−) and wild-type (Rho+/+) mice were subretinally injected with 2 µl of 2 × 1012 vp/ml AAVshQ1, which enables coexpression of shQ1 and EGFP in transduced retinal cells. (a) Expression of the 21-nucleotide (nt) shQ1 was confirmed by RNase protection assay in adult mice 10 days postinjection (n = 2). Protected RNA was separated on 15% denaturing polyacrylamide gels and detected using an shQ1 RNA probe, labeled with P32-γATP (lane Q1). In lane M, size markers indicate 10, 20, and 30 nt. Four weeks after AAVshNT (NT) and AAVshQ1 (Q1) administration at postnatal day 10, retinas were dissociated with trypsin and retinal cells sorted and analyzed by FACS (n = 4). (b) Representative plots of forward versus side scatter and histograms of EGFP fluorescence of the gated population (red dots on scatter plots) of NHR retinas are given for both AAVshNT (NT) and AAVshQ1 (Q1). (c) The bar chart indicates RHO mRNA expression from NHR mice in AAV-transduced (EGFP-positive) cells expressing shNT (NT) and shQ1 (Q1), isolated by FACS and quantified by qRT-PCR. (d) The bar chart indicates Rho mRNA expression from Rho+/+ mice in AAV-transduced (EGFP-positive) cells expressing shNT (NT) and shQ1 (Q1), isolated by FACS and quantified by qRT-PCR. Error bars represent SD values. *EGFP-positive fraction of cells; ***P < 0.001.

Article Snippet: The RHO-targeting short hairpin RNA shQ1 (target position nt 650–670; ref. 13 ) was cloned into pAAV-MCS (Stratagene, La Jolla, CA) with the EGFP reporter gene [pEGFP-1, BD Biosciences, Oxford, UK (Entrez Gene accession no. {"type":"entrez-nucleotide","attrs":{"text":"U57608","term_id":"1543069","term_text":"U57608"}} U57608 )] to create pAAVshQ1 ( ). shQ1 targets the RHO nucleotide sequence TCG TGT GGA ATC GAC TAC that differs from the mouse Rho sequence at four positions as underlined, TC A TG C GG G AT T GAC TAC, while encoding the same amino acids.

Techniques: In Vivo, Injection, Expressing, Rnase Protection Assay, Labeling, Fluorescence, Isolation, Quantitative RT-PCR

Journal:

Article Title: Improved Retinal Function in a Mouse Model of Dominant Retinitis Pigmentosa Following AAV-delivered Gene Therapy

doi: 10.1038/mt.2008.301

Figure Lengend Snippet: Viral spread in Pro347Ser mice. Fellow eyes from Pro347Ser (RHO347+/−Rho+/+) mice were injected with 1 µl of 2 × 1012 vp/ml AAVshQ1 and AAVshNT at postnatal day 10. Five weeks postinjection, eyes were fixed in 4% paraformaldehyde and retinal whole mounts prepared. Representative microscope images are depicted from eyes injected with AAVshNT (shNT; a) and AAVshQ1 (shQ1; b). Green label corresponds to EGFP fluorescence signal in the AAV-targeted region of the retina. Red dots border the extent of the retinas. Bar = 1 mm.

Article Snippet: The RHO-targeting short hairpin RNA shQ1 (target position nt 650–670; ref. 13 ) was cloned into pAAV-MCS (Stratagene, La Jolla, CA) with the EGFP reporter gene [pEGFP-1, BD Biosciences, Oxford, UK (Entrez Gene accession no. {"type":"entrez-nucleotide","attrs":{"text":"U57608","term_id":"1543069","term_text":"U57608"}} U57608 )] to create pAAVshQ1 ( ). shQ1 targets the RHO nucleotide sequence TCG TGT GGA ATC GAC TAC that differs from the mouse Rho sequence at four positions as underlined, TC A TG C GG G AT T GAC TAC, while encoding the same amino acids.

Techniques: Injection, Microscopy, Fluorescence