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Flourescent Genotyping
Working under the Program
for Genomic Applications (PGA), the flourescent genotyping unit
is involved in the development of consomic rat strains. They are
currently developing chromosome substution panels for FHHxBN as
well as SS/MCWxBN. With the use of the ABI 377 DNA Sequencer, the
team is capable of running 10 96-lane gels per week. This allows
them to investigate 120 animal genotypes using an average of 250
markers. The team aspires to eventually run 8 different markers
on one lane.
Radioactive Genotyping
Using P32 radiation genotyping the HMGC has the potential
to load 84 48-lane gels in a day (right). This enables the genotyping
team to canvass 4,032 genotypes per day and as much as 20,160 genotypes
per week.
Current projects
- Family Blood Pressure Project - The goals of the project are
to study the genetic contribution to hypertension in different
rat models. We have genetically mapped several quantitative trait
loci (QTLs) involved in blood pressure and other associated phenotypes
and are currently working on positional cloning efforts of two
of these QTL. Crosses and strains involved in this project are:
- GH x BN Study - A positional cloning project using congenic
animals having GH (Genetically Hypertensive) QTL regions introgressed
on the BN (Brown Norway) genomic background.
- Lyon Hypertension Study - Mapping of hypertension associated
phenotypes in a cross between the LH (Lyon Hypertensive) and
the LN (Lyon Normotensive), in collaboration with Dr. Jean
Sassard in Lyon, France. Also generating consomic lines introgressing
whole chromosomes (containing QTL determined in the mapping
cross) from the LH onto the genome of the BN control.
- SHR x BN Study - In a joint investigation with Dr. Jose
Krieger, the genotyping team has assisted in the development
of congenic strains for QTLs identified in a cross between
SHR (spontaneous hypertensive rat) and BN (Brown Norway) rats.
- Rf-1 Congenics Study - A project coordinated with the Netherlands.
- Genetic Control of BB Rat Autoimmunity - The goals of this project
are to determine the genetic factors causing type I diabetes in
the BB rat model, in collaboration with Dr Ake Lernmark at the
University of Washington. We have previously mapped several QTL
involved in type I diabetes. We are now involved in mapping another
locus as well as in developing congenic strains for positional
cloning and gene expression studies. Finally, in collaboration
with Dr. Hartmut Weiler, we are doing knockout studies of a candidate
gene in the mouse.
- Study of Ventilation and Sleep Behavior - The goals of the project
are to begin to define the magnitude and sites of genetic influence
on respiratory control at rest and with respiratory challenge.
A collaboration between Drs. Thom Feroah and Kingman Strohl (Case
Western Reserve Univ), the primary aims of the study are:
- To characterize the genetic transmission of traits of ventilation,
tidal volume, and frequency in an intercross between parental
animals from rat strains with high and low phenotypic values
at rest and when breathing is simulated by CO2.
- To identify chromosomal regions of interest involved in
ventilation by the use of novel and standard biometrical techniques
and an existing rat genetic linkage map.
Radiation Hybrid Mapping/Rat EST project
The use of radiation hybrids (RH) and RH maps has
revolutionized physical mapping of disease loci in human. The elegance
of this approach is in its simplicity; a single 96-well PCR reaction
can map a candidate gene with relatively high resolution. The construction
of a rat RH map is of great significance because the rat has been,
and continues to be, utilized as a powerful model system for investigating
human genetic diseases, particularly multifactorial diseases and
physiological traits. The goal of this project are to map rat ESTs
that are representative of a unique gene (UniGenes) onto RH framework
maps we have previously generated.
Rat cells carrying
a selectable marker are irradiated with a dose of X-rays to fragment
their chromosomes; the irradiated cells are fused with hamster cells,
and fusants are selected using the rat-derived marker. These cell
lines carry random fragments of rat DNA integrated into the hamster
chromosomes. Any particular fusion line carries only a small fraction
of the entire rat genome. As a result, genes which lie relatively
close to each other in the rat genome tend to be found simultaneously
in the same fusion cell line - much more often than expected by
chance. The closer together they lie, the more often they will occur
together: the frequency of co-occurrence is a measure of the physical
distance between the two genes. Relative frequencies of co-occurrence
can be used to order sets of three genes as well as to map gene
pairs.
 Radiation
hybrid mapping can be simply done, by performing PCR. Markers need
not be polymorphic in the rat, as long as they are distinguishable
from hamster homologs. The process can be automated from the initial
aliquoting of samples for PCR through gel reading and map construction.
The lab is capable of mapping about 700 genotypes a month.
Physical Mapping
 Concentrating
on the
Rf-1 study, the combined teams of Physical Mapping and Sequence
Analysis use a two-dimensional pool technique to classify and organize
genotypic data. This procedure is initiated with an Overgo (overlapping
oligonucleotides) Hybridization (protocol)
to high-density arrayed rat BAC (Bacterial Artificial Chromosome)
libraries. Then PCR is performed (protocol)
to confirm the hybridization data. This, however, only occurs after
growing up the BAC and purifying it (protocol).
The technique is then completed with a BAC-end sequencing. The combined
team can analyize a maximum of eight hybridizations per day. Using
five filters for each hybridization, the team can run a grand total
of forty films a day. The filters used have a typical coverage of
six, meaning that they should cover six BACs per marker. The current
project, Rf-1 congenics, requires the use of 450 markers or approximately
600 BACs.
SNPs and Sequencing
The reseach focuses on understanding how sequence variation in the
human genome predisposes to disease. Single nucleotide polymorphisms
(SNPs), one common form of sequence variation, occur frequently
throughout the human genome, and the DNA sequence of any two humans
differs in more than 2 million positions. While a large number of
these variants may have no discernible function, the laboratory
tries to explore whether some of these SNPs are associated with
incresed risk for disorders in humans. Furthermore, research focusses
on the evolution of these SNPs, and their relationship to each other.
Hopefully, results from this work will allow predictions about potential
functions of SNPs in the human genome.
Under NHLBI guidance as part of the Berkeley
PGA, the lab studies SNPs in genes affecting lipid metabolism,
a primary risk factor for cardiovascular disease in humans. Additional
projects focus on the role of SNPs in APP
(Amyloid Precursor Protein) and other genes in the development of
Alzheimer's Disease, the role of AML-1 in Accute Myloid Leukemia,
particularly in children with Down's Syndrome. Lastly, the lab explores
SNPs in genes such as CD36, Insulin Receptor, and PTPN1 to determine
their role in the development of Metabolic Syndrome, a disorder
causing obesity, hypertension, hyperlipidemia and insulin resistance
in humans.
To test associations of SNPs with these disorders
in large numbers of DNA samples from study cohorts of patients,
the lab utilizes the Invader® Assay for analyzing genetic
variation. The method has been developed by Third
Wave Technologies, a biotech firm based in Madison Wisconsin,
and the team collaborates with the company to explore new methods
for high-throughput SNP genotyping.
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