MRI detection of transcriptional regulation of gene expression in transgenic mice Batya Cohen, Keren Ziv, Vicki Plaks, Tomer Israely, Vyacheslav Kalchenko,

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MRI detection of transcriptional regulation of gene expression in transgenic mice Batya Cohen, Keren Ziv, Vicki Plaks, Tomer Israely, Vyacheslav Kalchenko, Alon Harmelin, Laura Benjamin, and Michal Neeman Derek Ju and John Kucharczyk December 3, 2010

Goal: noninvasive detection of reporter gene expression in live animals Current: Fluorescent, bioluminescent, and enzyme reporters for optical imaging and PET ▫High sensitivity ▫Low spatial and anatomical resolution Future: MRI detection of reporter genes ▫High 3D spatial and anatomical resolution ▫Multiple contrast mechanisms

Ferritin: the ideal reporter gene? Iron storage protein that is essential to life Ferritin substantially elevates R 2 in tissue ▫R 2 = transverse relaxation rate of water, 1/T 2 Linear dependence of R 2 on the magnetic field High R 2 at low iron loading

Experimental regulation of Ferritin Tissue specific VE cadherin promoter Tetracycline transactivator (tTA) tTA tetracyclin

Creation of dTG and sTG mice dTG=double transgenic sTG=single transgenic

Experiment 1:tTA controlled by VE- cadherin VE-cadherin is used in small percentage of cells  demonstrate sensitivity sTG : dTG :

Experiment 2: expression in utero VE-Cadherin expressed after day 13.5 High expression in the liver, heart and posterior, low in brain dTGsTG

Experiment 3:over expression in liver No difference between dTG and sTG EGFP demonstrated over expression of ferritin in dTG Liver maintains homeostasis of iron concentration sTGdTG

Experiment 4: dynamic changes in ferritin Induction of transgene with tetracycline withdrawal Unexpected: R2 drops with expression of ferritin Could be vacuoles sT G dTG

Conclusions Could detect expression in VE cells Could detect organ specific expression in embryos Ferritin regulation prevents detection in liver Dynamic changes in liver ferritin results in decreased R2

Uses in BE Tool for testing iron homeostasis MRI can be used to detect gene expression with high spatial and anatomical resolution