Journal: Scientific Reports

Article Title: Characterisation and use of a functional Gadd45g bacterial artificial chromosome

doi: 10.1038/s41598-018-35458-5

Figure Lengend Snippet: ( A ) Position of the mCherry reporter in the Gadd45g open reading frame of E15 BAC; ( B ) Detection of Cherry fluorescence in 11.5 dpc embryonic gonad. Green signal indicates location of PECAM. Blue is DAPI staining. ( C ) Cherry signal is detected in somatic cells of the gonad, which lack PECAM staining shown in ( D ). A few somatic cells (lacking PECAM) also lack Cherry signal (white arrows). ( E ) Lateral view of 10.5 dpc embryo showing Cherry fluorescence in developing neural tissue (forebrain (fb), midbrain (mb), hindbrain (hb)). ( F ) Dorsal view of same embryo reveals signal in neural tube (nt), trigeminal ganglion (tg) and facial ganglion (fg). ( G ) Section of embryo (in plane indicated by dotted line in ( E )) shows neural tube and dorsal root gangion (drg) fluorescence. Scale bar = 50 μm.

Article Snippet: A construct containing mCherry, nuclear localization signal and SV40pA terminator derived from Addgene plasmid GW1-Peredox-mCherry-NLS (#32381, a kind gift from Gary Yellen) was used to replace the rspL-neo cassette in a second round of transformation, restoring streptomycin resistance.

Techniques: Fluorescence, Staining

Journal: eLife

Article Title: AAV-Txnip prolongs cone survival and vision in mouse models of retinitis pigmentosa

doi: 10.7554/elife.66240

Figure Lengend Snippet: Figure 4. Effect of Txnip on ATP:ADP levels in retinitis pigmentosa (RP) cones in media with different carbon sources. (A) Representative ex vivo live images of PercevalHR-labeled cones in P20 rd1 retinas cultured with high-glucose, lactate-only, or pyruvate-only medium and transduced with Txnip (AAV8-RedO-Txnip, 1 109 vg/eye, plus AAV8-RO1.7-PercevalHR, 1 109 vg/eye) (RO1.7 is a shorter version of the red opsin [RedO] promoter with a similar expression pattern) or control (i.e., AAV8-RO1.7-PercevalHR, 1 109 vg/eye). Magenta: fluorescence by 405 nm excitation, indicating low-ATP: ADP; green: fluorescence by 488 nm excitation, indicating high-ATP:ADP. (B) Quantification of normalized PercevalHR fluorescence intensity ratio (FPercevalHR ex488nm: ex405nm, proportional to ATP:ADP ratio) in cones from P20 rd1 retinas in different conditions. The number in the square brackets ‘[]’ indicates the sample size, that is, the number of images taken from regions of interest of multiple retinas ( » 3 images per retina), in each condition. (C) Quantification of normalized PercevalHR fluorescence intensity of retinas infected with Txnip + siLdhb(#2) and Txnip + siNC in cones from P20 rd1 retina in lactate-only or pyruvate-only medium. (AAV8-RedO-Txnip, » 1 109 vg/eye; AAV8-RedO-shRNA » 1 109 vg/eye; plus AAV8-RO1.7-PercevalHR, 1 109 vg/eye.) (D) Representative ex vivo live images of PercevalHR-labeled cones in P20 rd1 retinas cultured in lactate-only medium, following transduction with Txnip.C247S (AAV8-RedO-Txnip.C247S, 1 109 vg/eye) or Txnip.S308A (AAV8-RedO-Txnip.S308A, 1 109 vg/eye). Magenta: fluorescence by 405 nm excitation, indicating low-ATP:ADP; green: fluorescence by 488 nm excitation, indicating high-ATP:ADP. (E) Quantification of normalized PercevalHR fluorescence intensity following transduction by Txnip, Txnip alleles, and control cones in P20 rd1 retinas cultured in lactate-only medium. Error bar: standard deviation. NS: not significant, p>0.05, **p<0.01, ***p<0.001, **** p< or <<0.0001. AAV: adeno-associated virus. The online version of this article includes the following figure supplement(s) for figure 4:

Article Snippet: DOI: https://doi.org/10.7554/eLife.66240 19 of 29 Continued Reagent type (species) or resource Designation Source or reference Identifiers Additional information Recombinant DNA reagent GFP-Txnip Addgene (Clark Distelhorst) 18758 PMID:16301999 Recombinant DNA reagent PercevalHR Addgene (Gary Yellen) 49082 PMID:24096541 Recombinant DNA reagent pAAV-RedO Botond Roska PMID:20576849 Recombinant DNA reagent pAAV-SynPVI (ProA7) Botond Roska PMID:31285614 Recombinant DNA reagent pAAV-SynP136 (ProA1) Botond Roska PMID:31285614 Software, algorithm RP cone counting This paper MATLAB scripts available at https://github.com/ sawyerxue/RP-cone-count Animals rd1 mice were the albino FVB strain, which carries the Pde6brd1 allele (MGI: 1856373).

Techniques: Ex Vivo, Labeling, Cell Culture, Transduction, Expressing, Control, Fluorescence, Infection, shRNA, Standard Deviation, Virus