SpringerProtocols: Abstract: Nitroreductase-Mediated Cell Ablation in Transgenic Zebrafish Embryos

Nitroreductase-Mediated Cell Ablation in Transgenic Zebrafish Embryos

By: Harshan Pisharath⁴, Michael J. Parsons⁴

Abstract

Full Text

Download PDF (280K)

Prodrug dependent cell ablation is a method that allows inducible and spatially restricted cell destruction. We describe transgenic methods to express the Escherichia coli nfsB in a tissue restricted manner in the zebrafish. This bacterial gene encodes a nitroreductase (NTR) enzyme that can render prodrugs such as metronidazole (Met) cytotoxic. Using the expression of NTR fused to a fluorescent protein, one can simultaneously make cells susceptible to prodrug treatment and visualize cell ablation as it occurs.

Affiliation(s): (4) Department of Surgery, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA

Book Title: Zebrafish: Methods and Protocols

Series: Methods in Molecular Biology | Volume: 546 | Pub. Date: Jan-01-2010 | Page Range: 133-143 | DOI: 10.1007/978-1-60327-977-2_9

Subject: Biochemistry

Key Words: Ablation - Nitroreductase - Metronidazole - Prodrug - Transactivation - Gal4 - UAS

References

Download References

1.	Yang, C. T., and Johnson, S. L. (2006). Small molecule-induced ablation and subsequent regeneration of larval zebrafish melanocytes, Development 133, 3563–3573.

2.	Borrelli, E., Heyman, R., Hsi, M., and Evans, R. M. (1988) Targeting of an inducible toxic phenotype in animal cells, Proc Natl Acad Sci U S A 85, 7572–7576.

3.	Clark, A. J., Iwobi, M., Cui, W., Crompton, M., Harold, G., Hobbs, S., Kamalati, T., Knox, R., Neil, C., Yull, F., and Gusterson, B. (1997). Selective cell ablation in transgenic mice expression E. coli nitroreductase, Gene Ther 4, 101–110.

4.	Drabek, D., Guy, J., Craig, R., and Grosveld, F. (1997). The expression of bacterial nitroreductase in transgenic mice results in specific cell killing by the prodrug CB1954, Gene Ther 4, 93–100.

5.	Smith, S. J., Kotecha, S., Towers, N., and Mohun, T. J. (2007). Targeted cell-ablation in Xenopus embryos using the conditional, toxic viral protein M2(H37A), Dev Dyn 236, 2159–2171.

6.	Poss, K. D., Keating, M. T., and Nechiporuk, A. (2003). Tales of regeneration in zebrafish, Dev Dyn 226, 202–210.

7.	Hu, L., Yu, C., Jiang, Y., Han, J., Li, Z., Browne, P., Race, P. R., Knox, R. J., Searle, P. F., and Hyde, E. I. (2003). Nitroaryl phosphoramides as novel prodrugs for E. coli nitroreductase activation in enzyme prodrug therapy, J Med Chem 46, 4818–4821.

8.	Johansson, E., Parkinson, G. N., Denny, W. A., and Neidle, S. (2003). Studies on the nitroreductase prodrug-activating system. Crystal structures of complexes with the inhibitor dicoumarol and dinitrobenzamide prodrugs and of the enzyme active form, J Med Chem 46, 4009–4020.

9.	Bridgewater, J. A., Springer, C. J., Knox, R. J., Minton, N. P., Michael, N. P., and Collins, M. K. (1995). Expression of the bacterial nitroreductase enzyme in mammalian cells renders them selectively sensitive to killing by the prodrug CB1954, Eur J Cancer 31A, 2362–2370.

10.	Cui, W., Gusterson, B., and Clark, A. J. (1999). Nitroreductase-mediated cell ablation is very rapid and mediated by a p53-independent apoptotic pathway, Gene Ther 6, 764–770.

11.	Cui, W., Allen, N. D., Skynner, M., Gusterson, B., and Clark, A. J. (2001). Inducible ablation of astrocytes shows that these cells are required for neuronal survival in the adult brain, Glia 34, 272–282.

12.	Isles, A. R., Ma, D., Milsom, C., Skynner, M. J., Cui, W., Clark, J., Keverne, E. B., and Allen, N. D. (2001). Conditional ablation of neurones in transgenic mice, J Neurobiol 47, 183–193.

13.	Felmer, R., Cui, W., and Clark, A. J. (2002). Inducible ablation of adipocytes in adult transgenic mice expressing the E. coli nitroreductase gene, J Endocrinol 175, 487–498.

14.	Kwak, S. P., Malberg, J. E., Howland, D. S., Cheng, K. Y., Su, J., She, Y., Fennell, M., and Ghavami, A. (2007). Ablation of central nervous system progenitor cells in transgenic rats using bacterial nitroreductase system, J Neurosci Res 85, 1183–1193.

15.	Wang, X. D., Shou, J., Wong, P., French, D. M., and Gao, W. Q. (2004). Notch1-expressing cells are indispensable for prostatic branching morphogenesis during development and re-growth following castration and androgen replacement, J Biol Chem 279, 24733–24744.

16.	Curado, S., Anderson, R. M., Jungblut, B., Mumm, J., Schroeter, E., and Stainier, D. Y. (2007). Conditional targeted cell ablation in zebrafish: a new tool for regeneration studies, Dev Dyn 236, 1025–1035.

17.	Pisharath, H., Rhee, J. M., Swanson, M. A., Leach, S. D., and Parsons, M. J. (2007). Targeted ablation of beta cells in the embryonic zebrafish pancreas using E. coli nitroreductase, Mech Dev 124, 218–229.

18.	Davison, J. M., Akitake, C. M., Goll, M. G., Rhee, J. M., Gosse, N., Baier, H., Halpern, M. E., Leach, S. D., and Parsons, M. J. (2007). Transactivation from Gal4-VP16 transgenic insertions for tissue-specific cell labeling and ablation in zebrafish, Dev Biol 304, 811–824.

19.	Fischer, J. A., Giniger, E., Maniatis, T., and Ptashne, M. (1988). GAL4 activates transcription in Drosophila, Nature 332, 853–856.

20.	Westerfield, M. (1993) The Zebrafish Book, University of Oregon, OR.

21.	Koster, R. W., and Fraser, S. E. (2001). Tracing transgene expression in living zebrafish embryos, Dev Biol 233, 329–346.

22.	Scheer, N., and Campos-Ortega, J. A. (1999). Use of the Gal4-UAS technique for targeted gene expression in the zebrafish, Mech Dev 80, 153–158.

23.	Kawakami, K., and Shima, A. (1999). Identification of the Tol2 transposase of the medaka fish Oryzias latipes that catalyzes excision of a nonautonomous Tol2 element in zebrafish Danio rerio, Gene 240, 239–244.

24.	Kawakami, K., Shima, A., and Kawakami, N. (2000). Identification of a functional transposase of the Tol2 element, an Ac-like element from the Japanese medaka fish, and its transposition in the zebrafish germ lineage, Proc Natl Acad Sci U S A 97, 11403–11408.

25.	Brand, A. H., and Perrimon, N. (1993). Targeted gene expression as a means of altering cell fates and generating dominant phenotypes, Development 118, 401–415.

26.	Duffy, J. B. (2002). GAL4 system in Drosophila: a fly geneticist’s Swiss army knife, Genesis 34, 1–15.

27.	O’Brien, B. A., Harmon, B. V., Cameron, D. P., and Allan, D. J. (1996). Beta-cell apoptosis is responsible for the development of IDDM in the multiple low-dose streptozotocin model, J Pathol 178, 176–181.

28.	Danial, N. N., and Korsmeyer, S. J. (2004). Cell death: critical control points, Cell 116, 205–219.

29.	Hughes, J., and Gobe, G. (2007). Identification and quantification of apoptosis in the kidney using morphology, biochemical and molecular markers, Nephrology (Carlton) 12, 452–458.

Comments (Loading...)

Post comment/View all comments