Page 56 - Mouse Molecular Genetics

Full Abstracts
Program number is above title. Author in bold is the presenter.
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of California, Davis, CA.
As part of KOMP2, an NIH Common Fund project to produce and phenotype 2,500 unique mutant mouse lines over a 5-year
period, we are applying RNAseq to characterize transcriptome changes in mutant tissues. We report here the RNAseq results
from 34 KOMP mutant lines derived from IKMC targeted stem cells (www.knockoutmouse.org ). Male homozygous (HOM)
mutant and wild-type (WT) control mice were ~50 3 days at tissue harvest. For each mutation, RNA extracted from ~ 4 RNA-
later stabilized or quick-frozen tissues from each of 5 male mice was used to create indexed cDNA libraries for sequencing on an
Illumina HiSeq DNA Analyzer. Approximately 4-5 libraries per lane produced ~20-25M ~45nt reads per library passing
trimming and QC which were mapped to the NCBI mouse transcriptome. Total read counts, from all alternative transcripts, were
compared between HOM and age-matched WT controls. Our RNAseq analysis revealed a high incidence of localized changes in
the 500kb region flanking the target gene. These regional changes were found in approximately 40% of libraries from mutants
produced from Velocigene ES cell clones, and from ~20% produced from CSD or EUCOMM targeted clones. We observed both
significant over- and under-expression of genes flanking the target. Some of these transcriptome changes are likely compensatory
and in response to physiological changes produced by the mutation. However, other changes could be due to disruption of
chromatin, introduction of foreign DNA causing localized silencing, and the insertion of an exogenous promoter causing
activation of neighboring genes. Global transcriptome changes were evaluated using clustering tools such as Gorilla, the NIAID
DAVID bioinformatics toolkit, and the Ingenuity package, in order to predict gene function based upon compensatory changes.
These data are available at
Supported by NIH grants U42OD011175 ,U54HG006364 and
U01HG004080.
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Coordinated activation of multiple tumor suppressor pathways in benign skin tumors identified by correlation analysis
of
Pten
gene expression. David Quigley
,
Ihn Young Song, Allan Balmain. Helen Diller Family Comprehensive Cancer Center,
University of California San Francisco, San Francisco, CA.
The DMBA/TPA mouse model of skin cancer creates numerous benign tumors in susceptible animals, a small percentage of
which progress to become invasive carcinomas. Most of these hyperplastic lesions remain benign after many months,
demonstrating that innate tumor suppression is effective even in an inflamed epithelium with an activating
H-ras
mutation. Most
studies of gene expression in tumors focus on detection of changes in levels of expression between two states. Here we have used
differential correlation analysis of gene expression between normal skin and benign skin tumors to investigate the rewiring of
potential tumor suppressor pathways at an early stage of carcinogenesis. Correlation was among the first statistical tools to be
applied to microarray data, but few studies have directly examined how changes in correlation after a tissue is perturbed can
elucidate changes in how genes work together under different biological conditions. We demonstrate that while the
Pten
gene
does not show significant changes in expression levels in benign tumors, it shows substantial changes in correlations with a
network of known tumor suppressors including
Tsc2
,
Smad4
and genes in the mTOR pathway which regulate translation and
growth. These relationships are not found in normal skin, and are lost during tumor progression from benign to malignant tumors.
We show by immunohistochemistry that Pten protein localizes to the cell membrane of keratinocytes in benign tumors but not
adjacent normal skin, compatible with an activation of Pten and consequent down-regulation of PI3K signaling. Transient
expression of oncogenic
Hras
in normal keratinocytes did not result in an up-regulation of Pten protein. Although we and others
have shown that mice heterozygous for
Pten
in their skin have increased susceptibility to skin tumors in this model, DMBA/TPA-
induced carcinomas in wild type mice do not lose
Pten
expression due to DNA copy number changes or mutation. This in vivo
finding would not be suggested using standard differential expression analysis or mutation screening. This work demonstrates a
framework for examining how pathway activation or suppression can be identified through a statistical approach which provides
important insights into gene expression network rewiring in tumors.
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A resource of vectors and ES cells for targeted deletion of microRNAs in mice. Haydn M Prosser
,
Hiroko Koike-Yusa,
Chukwuma A Agu, James D Cooper, Frances C Law, Allan Bradley. Wellcome Trust Sanger Institute, Cambridge, United
Kingdom.
The 21-23 nucleotide single-stranded RNAs classified as micro RNAs (miRNA) perform fundamental roles in a wide range of
cellular and developmental processes. MiRNAs regulate protein expression through sequence-specific base pairing with target
messenger RNAs (mRNA) reducing both their stability and the process of protein translation. At least 30% of protein coding
genes appear to be conserved targets for miRNAs. The miRNA genes are distributed throughout the genome either singly or in
clusters. We have generated a library of highly germ-line transmissible C57BL/6N mouse mutant embryonic stem (ES) cells with
targeted deletions within one miRNA allele for the majority of miRNA genes currently annotated within the miRBase registry.
These alleles have been designed to be highly adaptable research tools, as a variety of modifications of the targeted loci can be
achieved by applying the technique of recombinase mediated cassette exchange (RMCE). We demonstrate the use of this
approach for the reporter and conditional alleles. This ES cell resource can be searched electronically and is available from ES
cell repositories for distribution to the scientific community.