Abstract
Germline mutations of the breast tumor suppressor gene BRCA1 predispose women to breast and ovarian cancers. However, loss-of-function mutations of mouse Brca1 results in recessive embryonic lethality, which obscures the functions of BRCA1 in breast cancer formation. Cre-loxP-mediated tissue-specific knockout was employed to overcome this obstacle. We found that the presence of a ploxP-neo-loxP cassette in intron 10 of Brca1 resulted in severe interference with gene expression. The neo cassette was deleted in either embryonic stem cells or mice to generate the neo-less conditional knockout allele. Finally, we performed functional analysis of mammary tumorigenesis in Brca1 conditional knockout mice. The methods to generate and analyze these Brca1 conditional knockout mice are described in this chapter.
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References
Alberg, A. J. and Helzlsouer, K. J. (1997) Epidemiology, prevention, and early detection of breast cancer. Curr. Opin. Oncol. 9, 505–511.
Hill, A. D., Doyle, J. M., McDermott, E. W., and O’Higgins, N. J. (1997) Hereditary breast cancer. Br. J. Surg. 84, 1334–1339.
Brody, L. C. and Biesecker, B. B. (1998) Breast cancer susceptibility genes. BRCA1 and BRCA2. Medicine (Baltimore) 77, 208–226.
Paterson, J. W. (1998) BRCA1: a review of structure and putative functions. Dis. Markers 13, 261–274.
Lane, T. F., Deng, C., Elson, A., Lyu, M. S., Kozak, C. A., and Leder, P. (1995) Expression of Brca1 is associated with terminal differentiation of ectodermally and mesodermally derived tissues in mice. Genes Dev. 9, 2712–2722.
Miki, Y., Swensen, J., Shattuck-Eidens, D., et al. (1994) A strong candidate for the breast and ovarian cancer susceptibility gene BRCA1. Science 266, 66–71.
Xu, X., Weaver, Z., Linke, S. P., et al. (1999) Centrosome amplification and a defective G2-M cell cycle checkpoint induce genetic instability in BRCA1 exon 11 isoform-deficient cells. Mol. Cell 3, 389–395.
Deng, C. X. and Brodie, S. G. (2000) Roles of BRCA1 and its interacting proteins. Bioessays 22, 728–737.
Deng, C. X. (2002) Tumor formation in Brca1 conditional mutant mice. Environ. Mol. Mutagen. 39, 171–177.
Xu, X., Wagner, K. U., Larson, D., et al. (1999) Conditional mutation of Brca1 in mammary epithelial cells results in blunted ductal morphogenesis and tumour formation. Nat. Genet. 22, 37–43.
Deng, C., Wynshaw-Boris, A., Zhou, F., Kuo, A., and Leder, P. (1996) Fibroblast growth factor receptor 3 is a negative regulator of bone growth. Cell 84, 911–921.
Yang, X., Li, C., Xu, X., and Deng, C. (1998) The tumor suppressor SMAD4/DPC4 is essential for epiblast proliferation and mesoderm induction in mice. Proc. Natl. Acad. Sci. USA 95, 3667–3672.
Lakso, M., Pichel, J. G., Gorman, J. R., et al. (1996) Efficient in vivo manipulation of mouse genomic sequences at the zygote stage. Proc. Natl. Acad. Sci. USA 93, 5860–5865.
Wagner, K. U., Wall, R. J., St-Onge, L., et al. (1997) Cre-mediated gene deletion in the mammary gland. Nucleic Acids Res. 25, 4323–4330.
Xu, X., Li, C., Garrett-Beal, L., Larson, D., Wynshaw-Boris, A., and Deng, C. X. (2001) Direct removal in the mouse of a floxed neo gene from a three-loxP conditional knockout allele by two novel approaches. Genesis 30, 1–6.
Gu, H., Marth, J. D., Orban, P. C., Mossmann, H., and Rajewsky, K. (1994) Deletion of a DNA polymerase beta gene segment in T cells using cell type-specific gene targeting. Science 265, 103–106.
Nagy, A. (2000) Cre recombinase: the universal reagent for genome tailoring. Genesis 26, 99–109.
Hirotsune, S., Fleck, M. W., Gambello, M. J., et al. (1998) Graded reduction of Pafah1b1 (Lis1) activity results in neuronal migration defects and early embryonic lethality. Nat. Genet. 19, 333–339.
Nagy, A., Moens, C., Ivanyi, E., et al. (1998) Dissecting the role of N-myc in development using a single targeting vector to generate a series of alleles. Curr. Biol. 8, 661–664.
Partanen, J., Schwartz, L., and Rossant, J. (1998) Opposite phenotypes of hypomorphic and Y766 phosphorylation site mutations reveal a function for Fgfr1 in anteroposterior patterning of mouse embryos. Genes Dev. 12, 2332–2344.
Rucker, E. B., 3rd, Dierisseau, P., Wagner, K. U., et al. (2000) Bcl-x and Bax regulate mouse primordial germ cell survival and apoptosis during embryogenesis. Mol. Endocrinol. 14, 1038–1052.
Chen, L., Adar, R., Yang, X., Monsonego, E. O., Li, C., Hauschka, P. V., et al. (1999) Gly369Cys mutation in mouse FGFR3 causes achondroplasia by affecting both chondrogenesis and osteogenesis. J. Clin. Invest. 104, 1517–1525.
Li, C., Chen, L., Iwata, T., Kitagawa, M., Fu, X. Y., and Deng, C. X. (1999) A Lys644Glu substitution in fibroblast growth factor receptor 3 (FGFR3) causes dwarfism in mice by activation of STATs and ink4 cell cycle inhibitors. Hum. Mol. Genet. 8, 35–44.
Shen, S. X., Weaver, Z., Xu, X., et al. (1998) A targeted disruption of the murine Brca1 gene causes gamma-irradiation hypersensitivity and genetic instability. Oncogene 17, 3115–3124.
Brodie, S. G., Xu, X., Qiao, W., Li, W. M., Cao, L., and Deng, C. X. (2001) Multiple genetic changes are associated with mammary tumorigenesis in Brca1 conditional knockout mice. Oncogene 20, 7514–7523.
Weaver, Z., Montagna, C., Xu, X., et al. (2002) Mammary tumors in mice conditionally mutant for Brca1 exhibit gross genomic instability and centrosome amplification yet display a recurring distribution of genomic imbalances that is similar to human breast cancer. Oncogene 21, 5097–5107.
Liyanage, M., Coleman, A., du Manoir, S., et al. (1996) Multicolour spectral karyotyping of mouse chromosomes. Nat. Genet. 14, 312–315.
Mansour, S. L., Thomas, K. R., and Capecchi, M. R. (1988) Disruption of the proto-oncogene int-2 in mouse embryo-derived stem cells: a general strategy for targeting mutations to non-selectable genes. Nature 336, 348–352.
Meye, A., Wurl, P., Bache, M., et al. (2000) Colony formation of soft tissue sarcoma cells is inhibited by lipid-mediated antisense oligodeoxynucleotides targeting the human mdm2 oncogene. Cancer Lett. 149, 181–188.
Kaartinen, V. and Nagy, A. (2001) Removal of the floxed neo gene from a conditional knockout allele by the adenoviral Cre recombinase in vivo. Genesis 31, 126–129.
Wagner, K. U., McAllister, K., Ward, T., Davis, B., Wiseman, R., and Hennighausen, L. (2001) Spatial and temporal expression of the Cre gene under the control of the MMTV-LTR in different lines of transgenic mice. Transgenic Res. 10, 545–553.
Tybulewicz, V. L., Crawford, C. E., Jackson, P. K., Bronson, R. T., and Mulligan, R. C. (1991) Neonatal lethality and lymphopenia in mice with a homozygous disruption of the c-abl proto-oncogene. Cell 65, 1153–1163.
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Deng, CX., Xu, X. (2004). Generation and Analysis of Brca1 Conditional Knockout Mice. In: Schönthal, A.H. (eds) Checkpoint Controls and Cancer. Methods in Molecular Biology™, vol 280. Humana Press. https://doi.org/10.1385/1-59259-788-2:185
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DOI: https://doi.org/10.1385/1-59259-788-2:185
Publisher Name: Humana Press
Print ISBN: 978-1-58829-214-8
Online ISBN: 978-1-59259-788-8
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