Both the retroviral and electroporation-based models express very high levels of expression is driven by the Rosa26 promoter, which results in relatively weak expression in the brain (28). multiple whole-chromosome BLZ945 losses, particularly of chromosomes 8, 12 and 19. Analysis of murine and human CPC gene expression profiles and copy number changes revealed altered expression of genes involved in cell cycle, DNA damage response, and cilium function. High-throughput drug screening identified small molecule inhibitors that decreased the viability of CPC. These models will be valuable tools for understanding the biology of choroid plexus tumors and for testing novel approaches to therapy. Introduction Choroid plexus tumors are rare pediatric neoplasms that arise around the ventricles of the brain, and account for up to 20% of brain tumors in children under 1 year of age. These tumors can be divided into 3 subgroups based on histology: choroid plexus papillomas (CPPs, WHO grade I), atypical choroid plexus papillomas (aCPPs, WHO grade II) and choroid plexus carcinomas (CPCs, BLZ945 WHO grade III). CPPs have a favorable prognosis after surgical resection and rarely require additional treatment. CPCs, in contrast, usually require surgical removal followed by radiation and chemotherapy. Despite aggressive treatments, the 5-year overall survival rate is less than 60% (1,2) and the median progression-free survival (PFS) is only 13 months (3). Patients who do survive often suffer devastating side effects from the therapy, including neurocognitive deficits, endocrine disorders and secondary cancers. Effective treatments for CPC are lacking due to poor understanding of CPC biology and the paucity of patient specimens and animal models for studying the disease. The pathogenesis of choroid plexus tumors is not well understood. Mutations in the tumor suppressor gene are present in 50% of CPCs and have been associated with poor prognosis (4). However, whole genome sequencing of CPC patient specimens has not identified other recurrent single nucleotide variants, insertions/deletions, or focal copy number alterations (5). Rather, CPCs exhibit frequent chromosomal imbalances, with some tumors exhibiting multiple large chromosomal gains (hyperdiploid) and others showing predominantly large chromosomal losses (hypodiploid) (6-8). These studies suggest that copy number alterations might be oncogenic drivers of CPC. To understand the pathogenesis of CPC, we have created mouse models of hypodiploid Mouse monoclonal to IgG2b/IgG2a Isotype control(FITC/PE) CPC by activating the oncogene and inactivating the tumor suppressor in neural stem cells or progenitors. The resulting models are useful for understanding the biology of CPC and for testing novel therapies. Materials and Methods Animals Atoh1-Cre (B6.Cg-Tg(Atoh1-cre)1Bfri/J, stock number 01104) and p53LoxP (B6.129P2-Trp53tm1Brn/J , stock number 008462) mice, BLZ945 Nestin-Cre (B6.Cg-Tg(Nes-cre)1Kln/J, stock number 003771) and GFAP-Cre (FVB-Tg(GFAP-cre)25Mes/J, stock number 004600) were purchased from JAX. Blbp-Cre mice were purchased from NCI mouse repository (B6;CB-Tg(Fabp7-creLacZ)3Gtm/Nci, strain number: 01XM9). LSL-MycT58A mice were kindly provided by Rosalie Sears at Oregon Health and Science University. All animals were maintained in the animal facilities at SBP. All experiments were performed in accordance with national guidelines and regulations, and with the approval of the animal care and use committees at SBP, at the University of California San Diego (UCSD) and at St Jude Childrens research hospital. Histology and immunohistochemistry For histological analysis, animals were perfused with PBS followed by 4% paraformaldehyde (PFA). Brains were removed and fixed in 4% PFA overnight and then transferred to 70% ethanol and embedded in paraffin. Sections were then stained with hematoxylin and eosin or with Ki67 antibody (Abcam: ab15580) or MYC antibody (Abcam: ab32072). For immunofluorescent staining, BLZ945 brains from PFA-perfused animals were fixed overnight in 4% PFA, cryoprotected in 30% sucrose, frozen in Tissue Tek-OCT (Sakura Finetek), and cut into 12 m sagittal sections. Sections were blocked and permeabilized for 1 hr with PBS containing 0.1% Triton X-100 and 10% normal donkey serum, stained with primary antibodies (anti-Otx2: Millipore AB9566; anti-Aqp1: Santa Cruz SC-20810; anti-Ki67: Abcam ab15580; pH2A.X: Cell Signaling 9718P) overnight at 4C, and incubated with secondary antibodies for 1 hr at room temperature. Sections were counterstained with DAPI and mounted with Fluoromount-G (Southern Biotech) before being visualized using a Zeiss LSM 700 confocal microscope. Quantitative RT-PCR RNA was isolated using Qiagen RNeasy mini kit. Reverse transcription was done using iScript cDNA synthesis kit (Bio-Rad). Primers for qPCR are listed below. (forward: 5-ATGCCCCTCAACGTGAACTTC-3, reverse: BLZ945 5-CGCAACATAGGATGGAGAGCA-3); (forward: 5-CACAGCGTGGTGGTACCTTA-3, reverse: 5-TCTTCTGTACGGCGGTCTCT-3); (forward: 5-GAGATCGACTCTCTGTTCGAGG-3, reverse: 5-GCCCGTTGAAGAAGTCCTG-3) Viability assay Cells were plated in 384 well plates prior to drug treatment. 48 hours after treatment, cell viability was analyzed by CellTiter Glo assay (Promega) and results were collected on a Perkin Elmer Envision plate reader. Western blot Protein.