MRI detection of transcriptional regulation of gene expression in transgenic mice

Abstract

Ferritin, the iron storage protein, was recently suggested to be a candidate reporter for the detection of gene expression by magnetic resonance imaging (MRI). Here we report the generation of TET:EGFP-HAferritin (tet-hfer) transgenic mice, in which tissue-specific inducible transcriptional regulation of expression of the heavy chain of ferritin could be detected in vivo by MRI. We show organ specificity by mating the tet-hfer mice with transgenic mice expressing tetracycline transactivator (tTA) in liver hepatocytes and in vascular endothelial cells. Tetracycline-regulated overexpression of ferritin resulted in specific alterations of the transverse relaxation rate (R2) of water. Transgene-dependent changes in R2 were detectable by MRI in adult mice, and we also found fetal developmental induction of transgene expression in utero. Thus, the tet-hfer MRI reporter mice provide a new transgenic mouse platform for in vivo molecular imaging of reporter gene expression by MRI during both embryonic and adult life.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Generation of tet-hfer MRI reporter mice.
Figure 2: MRI detection of endothelium-selective overexpression of h-ferritin in the adult mouse brain.
Figure 3: In utero MRI detection of VE-cadherin mediated expression in E13.5 fetal liver and heart.
Figure 4: MRI detection of h-ferritin overexpression by liver hepatocytes.
Figure 5: Liver overexpression of h-ferritin in the liver-hfer mice.

Accession codes

Accessions

GenBank/EMBL/DDBJ

References

  1. 1

    Yang, M., Baranov, E., Moossa, A.R., Penman, S. & Hoffman, R.M. Visualizing gene expression by whole-body fluorescence imaging. Proc. Natl. Acad. Sci. USA 97, 12278–12282 (2000).

    CAS  Article  PubMed  Google Scholar 

  2. 2

    Contag, C.H. & Bachmann, M.H. Advances in in vivo bioluminescence imaging of gene expression. Annu. Rev. Biomed. Eng. 4, 235–260 (2002).

    CAS  Article  PubMed  Google Scholar 

  3. 3

    Ray, P., De, A., Min, J.J., Tsien, R.Y. & Gambhir, S.S. Imaging tri-fusion multimodality reporter gene expression in living subjects. Cancer Res. 64, 1323–1330 (2004).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  4. 4

    Louie, A.Y. et al. In vivo visualization of gene expression using magnetic resonance imaging. Nat. Biotechnol. 18, 321–325 (2000).

    CAS  Article  PubMed  Google Scholar 

  5. 5

    Yu, J., Liu, L., Kodibagkar, V.D., Cui, W. & Mason, R.P. Synthesis and evaluation of novel enhanced gene reporter molecules: detection of beta-galactosidase activity using 19F NMR of trifluoromethylated aryl beta-D-galactopyranosides. Bioorg. Med. Chem. 14, 326–333 (2006).

    CAS  Article  PubMed  Google Scholar 

  6. 6

    Li, Z. et al. Creatine kinase, a magnetic resonance-detectable marker gene for quantification of liver-directed gene transfer. Hum. Gene Ther. 16, 1429–1438 (2005).

    CAS  Article  PubMed  Google Scholar 

  7. 7

    Walter, G., Barton, E.R. & Sweeney, H.L. Noninvasive measurement of gene expression in skeletal muscle. Proc. Natl. Acad. Sci. USA 97, 5151–5155 (2000).

    CAS  Article  PubMed  Google Scholar 

  8. 8

    Koretsky, A.P., Lin, Y.J., Schorle, H. & Jaenisch, R. Genetic control of MRI contrast by expression of the transferrin receptor. Proc. Int. Soc. for Magn. Reson. in Med. 4, 69 (1996).

    Google Scholar 

  9. 9

    Moore, A., Josephson, L., Bhorade, R.M., Basilion, J.P. & Weissleder, R. Human transferrin receptor gene as a marker gene for MR imaging. Radiology 221, 244–250 (2001).

    CAS  Article  PubMed  Google Scholar 

  10. 10

    Alfke, H. et al. In vitro MR imaging of regulated gene expression. Radiology 228, 488–492 (2003).

    Article  PubMed  Google Scholar 

  11. 11

    Weissleder, R. et al. MR imaging and scintigraphy of gene expression through melanin induction. Radiology 204, 425–429 (1997).

    CAS  Article  PubMed  Google Scholar 

  12. 12

    Cohen, B., Dafni, H., Meir, G., Harmelin, A. & Neeman, M. Ferritin as an endogenous MRI reporter for noninvasive imaging of gene expression in C6 glioma tumors. Neoplasia 7, 109–117 (2005).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  13. 13

    Deans, A.E. et al. Cellular MRI contrast via coexpression of transferrin receptor and ferritin. Magn. Reson. Med. 56, 51–59 (2006).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  14. 14

    Genove, G., DeMarco, U., Xu, H., Goins, W.F. & Ahrens, E.T. A new transgene reporter for in vivo magnetic resonance imaging. Nat. Med. 11, 450–454 (2005).

    CAS  Article  PubMed  Google Scholar 

  15. 15

    Gilad, A.A et al. Artificial reporter gene providing MRI contrast based on proton exchange. Nat. Biotechnol. 25, 217–219 (2007).

    CAS  Article  PubMed  Google Scholar 

  16. 16

    Theil, E.C. Ferritin: at the crossroads of iron and oxygen metabolism. J. Nutr. 133, 1549S–1553S (2003).

    CAS  Article  PubMed  Google Scholar 

  17. 17

    Gottesfeld, Z. & Neeman, M. Ferritin effect on the transverse relaxation of water: NMR microscopy at 9.4 T. Magn. Reson. Med. 35, 514–520 (1996).

    CAS  Article  PubMed  Google Scholar 

  18. 18

    Vymazal, J., Brooks, R.A., Bulte, J.W., Gordon, D. & Aisen, P. Iron uptake by ferritin: NMR relaxometry studies at low iron loads. J. Inorg. Biochem. 71, 153–157 (1998).

    CAS  Article  PubMed  Google Scholar 

  19. 19

    Vymazal, J., Zak, O., Bulte, J.W., Aisen, P. & Brooks, R.A. T1 and T2 of ferritin solutions: effect of loading factor. Magn. Reson. Med. 36, 61–65 (1996).

    CAS  Article  PubMed  Google Scholar 

  20. 20

    Gossuin, Y., Muller, R.N. & Gillis, P. Relaxation induced by ferritin: a better understanding for an improved MRI iron quantification. NMR Biomed. 17, 427–432 (2004).

    CAS  Article  PubMed  Google Scholar 

  21. 21

    Wood, J.C., Fassler, J.D. & Meade, T. Mimicking liver iron overload using liposomal ferritin preparations. Magn. Reson. Med. 51, 607–611 (2004).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  22. 22

    Hardy, P.A. et al. Correlation of R2 with total iron concentration in the brains of rhesus monkeys. J. Magn. Reson. Imaging 21, 118–127 (2005).

    Article  PubMed  Google Scholar 

  23. 23

    Haacke, E.M. et al. Imaging iron stores in the brain using magnetic resonance imaging. Magn. Reson. Imaging 23, 1–25 (2005).

    CAS  Article  PubMed  Google Scholar 

  24. 24

    Bauminger, E.R. et al. Iron (II) oxidation and early intermediates of iron-core formation in recombinant human H-chain ferritin. Biochem. J. 296, 709–719 (1993).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  25. 25

    Ferreira, C. et al. Early embryonic lethality of H ferritin gene deletion in mice. J. Biol. Chem. 275, 3021–3024 (2000).

    CAS  Article  PubMed  Google Scholar 

  26. 26

    Thompson, K. et al. Mouse brains deficient in H-ferritin have normal iron concentration but a protein profile of iron deficiency and increased evidence of oxidative stress. J. Neurosci. Res. 71, 46–63 (2003).

    CAS  Article  PubMed  Google Scholar 

  27. 27

    Orino, K. et al. Ferritin and the response to oxidative stress. Biochem. J. 357, 241–247 (2001).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  28. 28

    Pham, C.G. et al. Ferritin heavy chain upregulation by NF-kappaB inhibits TNFalpha-induced apoptosis by suppressing reactive oxygen species. Cell 119, 529–542 (2004).

    CAS  Article  PubMed  Google Scholar 

  29. 29

    Giordani, A. et al. Contrasting effects of excess ferritin expression on the iron-mediated oxidative stress induced by tert-butyl hydroperoxide or ultraviolet-A in human fibroblasts and keratinocytes. J. Photochem. Photobiol. B 54, 43–54 (2000).

    CAS  Article  PubMed  Google Scholar 

  30. 30

    Kakhlon, O., Gruenbaum, Y. & Cabantchik, Z.I. Repression of the heavy ferritin chain increases the labile iron pool of human K562 cells. Biochem. J. 356, 311–316 (2001).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  31. 31

    Wilkinson IV, J. et al. Tissue-specific expression of ferritin H regulates cellular iron homeostasis in vivo. Biochem. J. 395, 501–507 (2006).

    Article  Google Scholar 

  32. 32

    Sun, J.F. et al. Microvascular patterning is controlled by fine-tuning the Akt signal. Proc. Natl. Acad. Sci. USA 102, 128–133 (2005).

    CAS  Article  PubMed  Google Scholar 

  33. 33

    Gory, S. et al. The vascular endothelial-cadherin promoter directs endothelial-specific expression in transgenic mice. Blood 93, 184–192 (1999).

    CAS  PubMed  Google Scholar 

  34. 34

    Kistner, A. et al. Doxycycline-mediated quantitative and tissue-specific control of gene expression in transgenic mice. Proc. Natl. Acad. Sci. USA 93, 10933–10938 (1996).

    CAS  Article  PubMed  Google Scholar 

  35. 35

    Cozzi, A. et al. Overexpression of wild type and mutated human ferritin H-chain in HeLa cells: in vivo role of ferritin ferroxidase activity. J. Biol. Chem. 275, 25122–25129 (2000).

    CAS  Article  PubMed  Google Scholar 

  36. 36

    Haacke, E.M., Xu, Y., Cheng, Y.C. & Reichenbach, J.R. Susceptibility weighted imaging (SWI). Magn. Reson. Med. 52, 612–618 (2004).

    Article  PubMed  Google Scholar 

  37. 37

    Carmeliet, P. & Collen, D. Molecular basis of angiogenesis. Role of VEGF and VE-cadherin. Ann. NY Acad. Sci. 902, 249–262 (2000).

    CAS  Article  PubMed  Google Scholar 

  38. 38

    Kim, I., Yilmaz, O.H. & Morrison, S.J. CD144 (VE-cadherin) is transiently expressed by fetal liver hematopoietic stem cells. Blood 106, 903–905 (2005).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  39. 39

    Grabill, C., Silva, A.C., Smith, S.S., Koretsky, A.P. & Rouault, T.A. MRI detection of ferritin iron overload and associated neuronal pathology in iron regulatory protein-2 knockout mice. Brain Res. 971, 95–106 (2003).

    CAS  Article  PubMed  Google Scholar 

  40. 40

    Kato, J. et al. A mutation, in the iron-responsive element of H ferritin mRNA, causing autosomal dominant iron overload. Am. J. Hum. Genet. 69, 191–197 (2001).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  41. 41

    Furth, P.A. et al. Temporal control of gene expression in transgenic mice by a tetracycline-responsive promoter. Proc. Natl. Acad. Sci. USA 91, 9302–9306 (1994).

    CAS  Article  PubMed  Google Scholar 

  42. 42

    Plaks, V., Kalchenko, V., Dekel, N. & Neeman, M. MRI analysis of angiogenesis during mouse embryo implantation. Magn. Reson. Med. 55, 1013–1022 (2006).

    Article  PubMed  Google Scholar 

  43. 43

    Sheehan, D.C. & Hrapchak, B.B. Theory and Practice of Histotechnology 2nd edn, 217 (Mosby, St Louis, 1980).

    Google Scholar 

Download references

Acknowledgements

We would like to acknowledge helpful discussions with A. Koretsky, P. Bendel, E. Keshet and Y. Dor. We would like to thank G. Damari and E. Ino for their help in generation and breeding of the transgenic mice. This work was supported by the Israel Science Foundation 391/02 and by the Minerva Foundation (to M.N.).

Author information

Affiliations

Authors

Contributions

B.C. generated the construct and derived the transgenic mice; K.Z., V.P. and M.N. performed the MRI experiments and conducted the data analyses; K.Z., V.P., B.C., T.I., V.K. and A.H. contributed to the ex vivo fluorescence and histological analysis, L.E.B. provided the VE-cadherin mice; M.N., B.C., K.Z. and V.P. wrote the manuscript and M.N. supervised the project.

Corresponding author

Correspondence to Michal Neeman.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Histological analysis of multiple organs in male VE cadherin h-fer mice. (PDF 136 kb)

Supplementary Fig. 2

Ferritin expression during embryonic development. (PDF 301 kb)

Supplementary Fig. 3

Prussian Blue staining of ferritin iron. (PDF 147 kb)

Supplementary Video 1

Contrast enhanced 3D gradient echo MRI of E13.5 pregnant mouse. The dataset was acquired at the end of the spin echo measurement of R2 relaxation, after intravenous administration of biotin-BSA-GdDTPA. Hyperintense areas show the maternal vasculature including maternal circulation in the placenta (same mouse as Figure 3). (AVI 885 kb)

Supplementary Video 2

Maximal intensity projection revealing the placenta of E13.5 pregnancy. Movie was derived from the 3D contrast enhanced gradient echo dataset of movie S3. (AVI 652 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Cohen, B., Ziv, K., Plaks, V. et al. MRI detection of transcriptional regulation of gene expression in transgenic mice. Nat Med 13, 498–503 (2007). https://doi.org/10.1038/nm1497

Download citation

Further reading

Search

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing