IUPS SYNTHESIUM
New Data Acquisition Technologies for Genome-Based Physiology
Andrew Greene Department of Physiology, Medical College of Wisconsin, Milwaukee,
USA
Physiological Genomics with High-throughput Phenotyping .
Anne Kwitek-Black Human and Molecular Genetics Center, Medical College of
Wisconsin, Milwaukee, USA
The Role of Rat in Functional Genomics: Not Just Big Mice.
Mark Rieder Department of Molecular Biotechnology, University of Washington,
Seattle, WA USA
Large-Scale Candidate Gene Polymorphism Analysis Using an Automated Laboratory
Discovery Pipeline.
Curt Sigmund Department of Internal Medicine and Physiology & Biophysics,
University of Iowa, Iowa City, USA
Approaches For High Throughput Generation of Transgenic Mice: Application to
Understanding Genetic Variation in The Genome.
The purpose of this session is to provide a review of a cross-section of the techniques,
tools and methods for developing unique animal models and then acquiring sequence and
physiological data in a high throughput manner. The speakers will present an overview
of the concepts of Physiological Genomics and provide insight into some of the
challenges facing this activity.
To obtain maximum utility of the Human Genome Project there is a critical need to
define the function of as many genes as possible, as rapidly as possible. Genes that
contribute to common disease are priorities. The speakers in this session will describe
several powerful approaches to dissect common multigenic diseases. In the overview
talk Dr. Greene will describe an NIH funded program designed to develop, phenotype,
and distribute chromosomal substitution strains of rats (consomic rat panels). The
program is designed to link biological functions of heart, lung, and blood systems to
genomic data, develop a renewable national resource for investigators to study the impact
of multiple disease genes on systems biology, and provide basic information that
investigators can use to understand the impact of allelic variance and their interactions
with the environment upon diseases that influence the heart, lung, and blood systems.
Environmental stressors such as hypoxia, exercise, and high salt intake will be used to
unmask deficiencies in normal homeostatic mechanisms and idiopathic mechanisms that
contribute to disease. Methods for the high-throughput phenotypic characterization of
theses strains of consomic rats will be described, in which approximately 300 phenotypes
specific to heart, lung, kidney, vasculature, and blood function in response to
environmental stressors (hypoxia, exercise, salt intake) are measured. This technique will
increase an understanding of the genetic basis of fundamental mechanistic pathways of
the heart, lung, kidney, blood, and vasculature responses to stress. The dissemination of
these data and resources to a large number of investigators through educational
workshops and national meeting symposiums will provide a valuable new tool to allow
the translation of genes to function and disease.
In the second talk Dr. Black will detail the utility of the rat as an important model for
physiological genomics. The rat was selected because it remains the dominant preclinical model system used by the NHLBI and by the pharmaceutical industry. Dr. Black
will describe the availability of resources for the rat and will outline the development of
panels of consomic rats. In a panel of consomic rats, a single chromosome is replaced one
at a time so that the contribution of genes on each chromosome can be assessed by
phenotyping the consomic strain for the traits of interest. Consomic rat panels enable one
to assess the contribution of genes specific to that chromosome. This is accomplished by
reducing the genetic differences in each test by providing inbred strains with a uniform
genetic background. Using comparative mapping strategies one can link these traits to the
genomes of the mouse and human.
In the third talk Dr. Rieder will discuss the practical aspects of high throughput
gene polymorphism analysis in human populations. This technique that relies on single
nucleotide substitutions, also known as single nucleotide polymorphisms (SNPs), is
currently being developed for use in further genetic analysis of complex traits such as
hypertension. Numerous technological advances have driven the study of human genetic
variation at the single basepair level. Most significant among these is the ability to
directly determine a DNA sequence biochemically. In addition, the introduction of
computerized methods for data acquisition has allowed the production of DNA sequence
data to parallel the exponential growth in computational power and driven the completion
of the Human Genome Project. The reduction in sequencing costs has allowed the
systematic analysis of candidate genes in multiple individuals to interrogate their
complete genetic variability. This ability provides a new starting point in which one is
armed with complete knowledge of the underlying population genetic structure of any
locus and allows for rational selection of highly informative genetic markers for use in
genotype-phenotype studies.
The development of high-throughput approaches for the characterization of
baseline human variation is rapidly emerging using highly automated fluorescence DNA
sequencer. Current instruments allow for near continuous operation with minimal handson preparation time using integrated robotics for automated sample handling and loading
onto a capillary electrophoresis array and a highly sensitive fluorescence-based detection
system for identifying labeled DNA fragments. This data acquisition technology can be
integrated with other robot stations for sample preparation prior to sequencing, and with
highly integrated database management systems to streamline the analysis of sequence
variation data. We have designed a laboratory discovery pipeline, which allows for the
routine analysis of up to 25 kb of baseline genomic sequence/week for SNP discovery in
a sample of 48 individuals from two diverse populations. This technology is providing a
platform for the discovery of large numbers of SNPs that can be applied to
genotype/phenotype analysis. Also, the determination of SNP combinations occurring on
the same maternally or paternally derived chromosome defines haplotypes which may
permit the application of cladistic-based approaches in the analysis of quantitative traits.
Finally in the last talk Dr. Sigmund will discuss the use of transgenic mice as a tool to
explore the impact of genetic variation on phenotype. Dr. Sigmund will describe his
system to examine the physiological consequences of genetic variation using a high
throughput gene targeting system. In this system, identical genes, differing only in
specific variants identified in human populations, are targeted in a single copy to an
identical locus in the genome. Gene targeting is used because classical transgenic
approaches have significant weaknesses that prevent their use in this context. Using
classical transgenic approaches, transgenic founders that arise from a single construct all
differ with respect to both the insertion site and the transgene copy number, both of
which can impart strong effects on the pattern and magnitude of gene expression. Using a
unique approach transgenes can be inserted such that only a single copy will be expressed
at an identical insertion site. The method relies on the use of a specific ES cell line
termed BK4 which was previously manipulated to delete a portion of the hypoxanthine
phosphoribosyl transferase HPRT gene.
This talk will describe the use this system to examine the physiological consequences of
genetic variation in the angiotensinogen gene. Data will be presented that shows the high
efficiency of homologous recombination at the HPRT locus, and the appropriate tissueand cell-specific expression of transgenes integrated upstream of the HPRT locus .
Discussion will focus on the pros and cons of using the HPRT locus as a target for
transgenes and for generating transgenic mice with high throughput.