MRI detection of transcriptional regulation of gene expression in transgenic mice

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 3D spatial and anatomical resolution▫Multiple contrast mechanisms

Ferritin: the ideal reporter gene?

•Iron storage protein that is essential to life

•Ferritin substantially elevates R2 in tissue▫R2 = transverse relaxation

rate of water, 1/T2

•Linear dependence of R2 on the magnetic field

•High R2 at low iron loading

Experimental regulation of Ferritin

Tissue specific VE cadherin promoter

Tetracycline transactivator (tTA)

tTAtetracyclin

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

dTG

sTG

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

concentrationsTG dTG

Experiment 4: dynamic changes in ferritin

•Induction of transgene with tetracycline withdrawal

•Unexpected: R2 drops with expression of ferritin

•Could be vacuoles

sTG

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