Radiation Hybrid Maps of Medaka Chromosomes LG 12, 17, and 22

The Medaka is an excellent genetic system for studies of vertebrate development and disease and environmental and evolutionary biology studies. To facilitate the mapping of markers or the cloning of affected genes in Medaka mutants identified by forward-genetic screens, we have established a panel of whole-genome radiation hybrids (RHs) and RH maps for three Medaka chromosomes. RH mapping is useful, since markers to be mapped need not be polymorphic and one can establish the order of markers that are difficult to resolve by genetic mapping owing to low genetic recombination rates. RHs were generated by fusing the irradiated donor, OLF-136 Medaka cell line, with the host B78 mouse melanoma cells. Of 290 initial RH clones, we selected 93 on the basis of high retention of fragments of the Medaka genome to establish a panel that allows genotyping in the 96-well format. RH maps for linkage groups 12, 17, and 22 were generated using 159 markers. The average retention for the three chromosomes was 19% and the average break point frequency was ∼33 kb/cR. We estimate the potential resolution of the RH panel to be ∼186 kb, which is high enough for integrating RH data with bacterial artificial chromosome clones. Thus, this first RH panel will be a useful tool for mapping mutated genes in Medaka

Characterization of Ovarian Cancer Ascites on Cell Invasion, Proliferation, Spheroid Formation, Gene Expression in an In Vitro Model of Epithelial Ovarian Cancer

At least one third of all cases of epithelial ovarian cancer are associated with the production of ascites, although its effect on tumor cell microenvironment remains poorly understood. This study addresses the effect of the heterologous acellular fraction of ovarian cancer-derived ascites on a cell line (OV-90) derived from the chemotherapy-naive ovarian cancer patient. Ascites were assayed for their effect on cell invasion, growth, spheroid formation. When compared to either no serum or 5% serum, ascites fell into one of two categories: stimulatory or inhibitory. RNA from OV-90 cells exposed to selected ascites were arrayed on an Affymetrix HG-U133A GeneChip. A supervised analysis identified a number of differentially expressed genes, quantitative polymerase chain reaction validation based on OV-90 cells exposed to 54 independent ascites demonstrated that stimulatory ascites affected the expression of ISGF3G, TRIB1, MKP1, RGS4, PLEC1, MOSPD1 genes. In addition, TRIB1 expression was shown to independently correlate with prognosis when its expression was ascertained in an independent set of primary cultures established from ovarian ascites. The data support the validity of the strategy to uncover molecular events that are associated with tumor cell behavior, highlight the impact of ascites on the cellular, molecular parameters of ovarian cancer

Effect of Ovarian Cancer Ascites on Cell Migration and Gene Expression in an Epithelial Ovarian Cancer In Vitro Model1

A third of patients with epithelial ovarian cancer (EOC) present ascites. The cellular fraction of ascites often consists of EOC cells, lymphocytes, and mesothelial cells, whereas the acellular fraction contains cytokines and angiogenic factors. Clinically, the presence of ascites correlates with intraperitoneal and retroperitoneal tumor spread. We have used OV-90, a tumorigenic EOC cell line derived from the malignant ascites of a chemonaive ovarian cancer patient, as a model to assess the effect of ascites on migration potential using an in vitro wound-healing assay. A recent report of an invasion assay described the effect of ascites on the invasion potential of the OV-90 cell line. Ascites sampled from 31 ovarian cancer patients were tested and compared with either 5% fetal bovine serum or no serum for their nonstimulatory or stimulatory effect on the migration potential of the OV-90 cell line. A supervised analysis of data generated by the Affymetrix HG-U133A GeneChip identified differentially expressed genes from OV-90 cells exposed to ascites that had either a nonstimulatory or a stimulatory effect on migration. Ten genes (IRS2, CTSD, NRAS, MLXIP, HMGCR, LAMP1, ETS2, NID1, SMARCD1, and CD44) were upregulated in OV-90 cells exposed to ascites, allowing a nonstimulatory effect on cell migration. These findings were validated by quantitative polymerase chain reaction. In addition, the gene expression of IRS2 and MLXIP each correlated with prognosis when their expression was assessed in an independent set of primary cultures established from ovarian ascites. This study revealed novel candidates that may play a role in ovarian cancer cell migration

The LN54 Radiation Hybrid Map of Zebrafish Expressed Sequences

To increase the density of a gene map of the zebrafish, Danio rerio, we have placed 3119 expressed sequence tags (ESTs) and cDNA sequences on the LN54 radiation hybrid (RH) panel. The ESTs and genes mapped here join 748 SSLp markers and 459 previously mapped genes and ESTs, bringing the total number of markers on the LN54 RH panel to 4226. Addition of these new markers brings the total LN54 map size to 14,372 cR, with 118 kb/cR. The distribution of ESTs according to linkage groups shows relatively little variation (minimum, 73; maximum, 201). This observation, combined with a relatively uniform size for zebrafish chromosomes, as previously indicated by karyotyping, indicates that there are no especially gene-rich or gene-poor chromosomes in this species. We developed an algorithm to provide a semiautomatic method for the selection of additional framework markers for the LN54 map. This algorithm increased the total number of framework markers to 1150 and permitted the mapping of a high percentage of sequences that could not be placed on a previous version of the LN54 map. The increased concentration of expressed sequences on the LN54 map of the zebrafish genome will facilitate the molecular characterization of mutations in this species