MRI detection of transcriptional regulation of gene expression

Goal: noninvasive detection of reporter gene expression in live animals • Current: Fluorescent, bioluminescent,

Ferritin: the ideal reporter gene? • Iron storage protein that is essential to life

Experimental regulation of Ferritin t. TA tetracyclin Tissue specific VE cadherin promoter Tetracycline transactivator

Creation of d. TG and s. TG mice • d. TG=double transgenic • s.

Experiment 1: t. TA controlled by VEcadherin • VE-cadherin is used in small percentage

Experiment 2: expression in utero • VE-Cadherin expressed after day 13. 5 • High

Experiment 3: over expression in liver • No difference between d. TG and s.

Experiment 4: dynamic changes in ferritin • Induction of transgene with tetracycline withdrawal •

Conclusions • Could detect expression in VE cells • Could detect organ specific expression

Uses in BE • Tool for testing iron homeostasis • MRI can be used

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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 20. 309 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 3 D 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 t. TA tetracyclin Tissue specific VE cadherin promoter Tetracycline transactivator (t. TA)

Creation of d. TG and s. TG mice • d. TG=double transgenic • s. TG=single transgenic

Experiment 1: t. TA controlled by VEcadherin • VE-cadherin is used in small percentage of cells demonstrate sensitivity s. TG : d. TG :

Experiment 2: expression in utero • VE-Cadherin expressed after day 13. 5 • High expression in the liver, heart and posterior, low in brain s. TG d. TG

Experiment 3: over expression in liver • No difference between d. TG and s. TG • EGFP demonstrated over expression of ferritin in d. TG • Liver maintains homeostasis of iron concentration s. TG d. TG

Experiment 4: dynamic changes in ferritin • Induction of transgene with tetracycline withdrawal • Unexpected: R 2 drops with expression of ferritin • Could be vacuoles s. T G d. TG

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 R 2

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