Transcript PPT - Department of Biology

Microarray analysis identifies keratin loci as
sensitive biomarkers for thyroid hormone
disruption in salamanders (Ambystoma)
Department of Biology and
Amphibian Growth Project
THE GOAL
To begin characterization of gene expression
during salamander metamorphosis
Hypothesis: no variation in gene expression
during metamorphosis
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
Robert B. Page, James R. Monaghan, Amy
K. Samuels, Jeramiah J. Smith, and
Christopher K. Beachy and S. Randal Voss
HOW A MICROARRAY WORKS
theoretically…
For comparison to endocrine
disrupted metamorphosis
To identify loci that will
be useful in field
diagnostics of normal
and endocrine
disrupted
metamorphosis
To correlate patterns of
gene expression with
morphological,
developmental, and life
history malformations
during endocrine disruption
THE PLAN
Basically…..
Grow axolotls (Ambystoma mexicanum)
Skin
Collect
cDNA
MICROARRAY
Liver
mRNA
brain
For our microarrays, we use Affymetrix GeneChips to
quantify gene expression for approximately 4800
genes, many of which are homologous to human
genes. This allows for a comparison to the human
genome, and establishes salamander as an excellent
model system to understand the role of xenobiotics in
human health issues.
…and an example of ours (based
on the animal on the above cover!)
28 days after TH immersion
epithelial membrane protein 1
is upregulated in this
metamorphosing salamander
There are approximately 4800 genes for
examination on this GeneChip (or “microarray”).
This work is funded by
SOME OF OUR
MICROARRAY RESULTS
GREEN IS “OFF GENES”; RED
IS “ON” GENES.
Genes listed here are those
that were significant at P <
0.001, exhibited at least a
two-fold change in expression
when compared to controls,
and also have a match to the
human genome. There are
many other significant
patterns of gene expression
that do not meet these three
criteria and are subject to
further analysis.
PCR verification
of microarray
AND OUR EXPERIMENT
IMMERSION
CONTROL
IN T4
0 days
2 days
12 days
28 days
21 animals (3 per treatment), thus 21
GeneChips used for analysis.
Department of Biology and Salamander
Genome Project
We used PCR methods as a secondary verification of
our microarray results. We chose to examine keratin
gene expression; these genes were an important part of
the identified genes presented above and should be
ideal for field diagnostics of endocrine-disrupted
metamorphic development (see below). Red arrows
indicate significant up-regulated metamorphic genes
(“turned on genes”) and green arrows indicated downregulated metamorphic genes (“turned off genes”).
This is a “housekeeping” gene, i.e, it is expressed at the same levels
all the time and is used as a control and baseline to evaluate up- and
down-regulation of the other PCR products.
This work succeeds because of the continued
work of the members of the Amphibian Growth
Project: Drew Henry, Janel Richter, Ken Cabarle,
Claude Ouedraego, Jeremiah Johnson, Heather
Modrow, Judd Entzel and (especially) Karen
Pocha-Melby.
COMPARING THE STANDARD Gene
Expression Profile (GEP) WITH AN
ENDOCRINE DISRUPTED GEP
0 days
2 days
12 days
28 days
Atrazine dosage
0 g/L (ppb)
1 g/L (ppb)
5 g/L (ppb)
The establishment of the
gene expression profile
during normal induction of
metamorphosis now
allows for a comparison to
endocrine-disrupted
metamorphosis. This
project is ongoing; please
visit this experiment at the
AGP in Moore 232 and
214.