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ABSTRACT: Cre-loxp mediated conditional knockout strategy has played critical roles for revealing functions of many genes essential for development, as well as the causal relationships between gene mutations and diseases in the postnatal adult mice. One key factor of this strategy is the availability of mice with tissue- or cell type-specific Cre expression. However, the success of the traditional molecular cloning approach to generate mice with tissue specific Cre expression often depends on luck. Here we provide a better alternative by using bacterial artificial chromosome (BAC)-based recombineering to insert iCreERT2 cDNA at the ATG start of the Upk2 gene. The BAC-based transgenic mice express the inducible Cre specifically in the urothelium as demonstrated by mRNA expression and staining for LacZ expression after crossing with a Rosa26 reporter mouse. Taking into consideration the size of the gene of interest and neighboring genes included in a BAC, this method should be widely applicable for generation of mice with tissue specific gene expression or deletions in a more specific manner than previously reported.
PLoS ONE 01/2012; 7(4):e35243. · 4.09 Impact Factor
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Orit Karni-Schmidt,
Mireia Castillo-Martin, Tian Huai Shen,
Tian HuaiShen,
Nataliya Gladoun,
Josep Domingo-Domenech,
Marta Sanchez-Carbayo,
Yingchun Li,
Scott Lowe,
Carol Prives,
Carlos Cordon-Cardo
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ABSTRACT: The TP63 gene, a member of the TP53 tumor suppressor gene family, can be expressed as at least six isoforms due to alternative promoter use and alternative splicing. The lack of p63 isoform-specific antibodies has limited the analysis of the biological significance of p63. We report a novel set of well-defined antibodies to examine p63 isoforms in mouse and human urothelium during embryogenesis and tumor progression, respectively. We provide evidence that basal and intermediate urothelial cells express p63 isoforms, with the TAp63 variant the first to be detected during development, whereas umbrella cells are characterized by a p63-negative phenotype. Notably, we report that p63-null mice develop a bladder with an abnormal urothelium, constituted by a single layer of cells that express uroplakin II and low molecular weight cytokeratins, consistent with an umbrella cell phenotype. Finally, analysis of 202 human bladder carcinomas revealed a new categorization of invasive tumors into basal-like (positive for ΔNp63 and high molecular weight cytokeratins and negative for low molecular weight cytokeratins) versus luminal-like (negative for ΔNp63 and high molecular weight cytokeratins and positive for low molecular weight cytokeratins) phenotypes, with ΔNp63 expression associated with an aggressive clinical course and poor prognosis. This study highlights the relevance of p63 isoforms in both urothelial development and bladder carcinoma progression, with ΔNp63 acting as an oncogene in certain invasive bladder tumors.
American Journal Of Pathology 03/2011; 178(3):1350-60. · 4.89 Impact Factor
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ABSTRACT: Although bladder cancer represents a serious health problem worldwide, relevant mouse models for investigating disease progression or therapeutic targets have been lacking. We show that combined deletion of p53 and Pten in bladder epithelium leads to invasive cancer in a novel mouse model. Inactivation of p53 and PTEN promotes tumorigenesis in human bladder cells and is correlated with poor survival in human tumors. Furthermore, the synergistic effects of p53 and Pten deletion are mediated by deregulation of mammalian target of rapamycin (mTOR) signaling, consistent with the ability of rapamycin to block bladder tumorigenesis in preclinical studies. Our integrated analyses of mouse and human bladder cancer provide a rationale for investigating mTOR inhibition for treatment of patients with invasive disease.
Genes & development 04/2009; 23(6):675-80. · 12.08 Impact Factor
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ABSTRACT: PML nuclear bodies (NBs) are nuclear structures that have been implicated in processes such as transcriptional regulation, genome stability, response to viral infection, apoptosis, and tumor suppression. PML has been found to be essential for the formation of the NBs, as these structures do not form in Pml null cells, although PML add back fully rescues their formation. However, the basis for such a structural role of PML is unknown. We demonstrate that PML contains a SUMO binding motif that is independent of its SUMOylation sites and is surprisingly necessary for PML-NB formation. We demonstrate that the PML RING domain is critical for PML SUMOylation and PML-NB formation. We propose a model for PML-NB formation whereby PML SUMOylation and noncovalent binding of PML to SUMOylated PML through the SUMO binding motif constitutes the nucleation event for subsequent recruitment of SUMOylated proteins and/or proteins containing SUMO binding motifs to the PML NBs.
Molecular Cell 12/2006; 24(3):331-9. · 14.18 Impact Factor
