Download Detailed Exercises

PhysGen hands-on exercises
FALL 2007
These exercises are designed to make the user familiar with how to view and analyze the
genotyping and phenotype data for the rat strains developed by PhysGen
(http://pga.mcw.edu). The main page contains links to a variety of data for the consomic rat
strains and the new ENU mutant strains, including strain profiles, phenotyping, and
genotyping data.
From the home page (http://pga.mcw.edu), the data and other information about the rat
strains are grouped by “consomic” and “ENU mutant” to distinguish the different rat
strains and phenotyping protocols used to physiologically characterize the rats. Details about
the phenotyping protocols can be found under the “Protocols” link for both Consomic and
ENU Mutant rat strains found under “General Info” on the home page.
The first exercises (#1-2) highlight the new ENU mutant strain results – both genotyping and
phenotyping. For the ENU strains, the screening protocol provides results for 84 phenotypic
measurements of heart, lung, vascular, renal and blood measurements. All measurements
are made in each rat over a three week period.
Question 1: What mutant rat strains are available? Do they have any interesting traits?
Answer: The list of target genes for the ENU mutant rat strains is available on the main page
(http://pga.mcw.edu). A link to the KO rats under the PhysGen stories brings up information
about the targeted gene list as well as currently available mutant strains. Detailed
information about each mutant strain is available through Data→ ENU Mutant→
Genotype. The Allele Symbol column provides a link to the Rat GBrowser on the Rat
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Genome Database website (http://rgd.mcw.edu). A gene report is available for each ENU
strain which includes a description of function and biological process.
A new phenotyping strategy is being used for the ENU mutant strains. In addition to the
ENU mutant strains, the parental strains (SS, BN, FHH) and some consomic strains have
been screened using this new screening protocol. The results for the parental strains (SS,
BN, FHH) are available under Data→ ENU Mutant→ Phenotype. The results for the
consomic rat strains studied using the screening protocol can be found under Data →
Consomic → Second Screen. This database is updated quarterly as new strains are
completed.
Question 2: Is the level of plasma cholesterol altered in any of the ENU mutant strains on
the FHH background?
Answer:
The plasma cholesterol is
reduced in both male and
female FHH-Lcatm1Mcwi. These
results are shown in the graph
to the right (red arrows and
red box). The results for the
male and female FHH
parentals are the blue bars.
♂♀
The following exercises will
use the phenotyping results for the consomic rat panels found under the “Phenotype” link
under “Consomic” on the main page (http://pga.mcw.edu).
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Question 3: Which BN chromosomes, when introgressed onto a Dahl Salt-sensitive (SS)
background, confer protection against left ventricular infarct following global cardiac
ischemia? Does gender or stressor (chronic hypoxia) influence this protection? Is the
response the same in rats with a Fawn-Hooded Hypertensive (FHH) background?
Hint: Use the Cardiac protocol results to find the left ventricular infarct size phenotype.
Answer: Under normoxic conditions, the male BN, SS-6BN, SS-2BN and SS-7BN rats have a
significantly reduced infarct size compared to normoxic male SS rats following global
ischemia. Following 2 weeks of chronic hypoxia (12% O2), the male BN, SS-2BN, SS-7BN
were protected from global cardiac ischemia compared to the hypoxic male SS rats. The
female BN, SS-2BN, and SS-6BN rats had similar responses to the male rats under both
chronic hypoxia and normoxia.
Question 4: Are there any SSBN consomic strains that do not develop hypertension
following a high salt diet? If so, are males and females similar in their response? How else
do these strains differ from the parental SS strain?
Hint: Use the Renal B protocol to find the mean arterial blood pressure results. Use the
Strain Profile tool to learn more about individual strains.
Answer: In addition to the BN strain (male and female), the SS-5BN, SS-18BN, SS-13BN and
SS-7BN males have significantly lower mean arterial blood pressure following a high salt diet
compared to the SS males. In the females, only the BN and SS-5BN rats have a lower mean
arterial pressure compared to female SS rat on a high salt diet. The strain profile for the
SS-5BN rats provides a list of other phenotypes from all protocols whereby the SS-5BN differs
significantly from the SS rats including other Renal (microalbumin and protein excretion) and
Vascular (relaxation response to acetylcholine) phenotypes measured after a high salt diet
for several weeks.
Question 5: Does a chronic hypoxia affect right ventricular hypertrophy in both male and
female rats in a similar manner?
Hint: The Lung protocol has the right ventricle/left ventricle weight ratio phenotype.
Answer: Some strains have a similar response in both genders while other strains are quite
different. For example, the SD male and female rats are not significantly different from one
another, while the WKY male rats have a significant right ventricular hypertrophy compared
to the female WKY rats.
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PhysGen hands-on exercises
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Step by step instructions to get answers to questions 2-5:
Question 2:
1. Begin at the PhysGen home page (http://pga.mcw.edu) and select link to “Phenotype”
under Data → ENU Mutant.
2. From the Phenotype Data Status screen, click on the radio button next to Biochemistry
protocol and then click “Visualization & Statistics” button.
3. From the Rat Select Option page, choose the FHH ENU strains, include both genders,
highlight plasma cholesterol (phenotype #9), click “Do analysis” button on bottom of
page.
Question 3:
1. Go to PhysGen home page (http://pga.mcw.edu), click on link to “Phenotype” under
“Consomic” data. Select Cardiac protocol (for left ventricular infarct size phenotype),
click on “Visualization & Statistics” button.
2. User must make several choices on this page to get phenotype data results. First,
choose SSBN consomic rats (other choices are Parental strains and FHBN consomics).
Atmospheric conditions should be “both”. “Diet condition” only has one choice for this
protocol (0.4% salt). “Gender” should be “both” in this case. Under Select phenotypes,
Scroll down to highlight “post-ischemic infarct size (%LV). Finally, click “Do Analysis”
button for results.
3. This produces a large graph with the mean results for all male and female strains under
both normoxia and hypoxia plotted as individual bars from the lowest to highest
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response. To determine if any of the consomic strains differ significantly from the
parental strains (BN & SS), select the “check all strains” button found below the graph
and summary table. Next click on the “ANOVA analysis” button.
4. Four summary graphs should appear (hypoxic males, hypoxic females, normoxic males,
normoxic females) with the parental strains highlighted in blue (SS) and red (BN) bars
and all consomic strains in grey. Strain information (strain, atmospheric condition, diet,
gender, mean value for phenotype) appears when the mouse is moved over each bar.
5. To determine if there are significant differences among strains from these sub-groups of
data (e.g. hypoxic males), review the table below the graph to see the analysis performed
and the corresponding results. For the hypoxic male SSBN consomic rats for left
ventricular infarct size, the non-parametric ANOVA results indicate that there is a
statistically significant difference. To get post-hoc results, use the non-parametric
Dunnett’s test option, highlight the strain you wish to compare against (e.g. SS, 12% O 2,
male) and select button on right (non-parametric Dunnett test) for results.
6. Another graph appears (see graph below) with the strain selected for comparison (male,
hypoxic SS) in dark grey, strains that are significantly different than the selected strain
appear red and strains not significantly different than the selected strain are light grey.
The results from this example indicate that the BN, SS-2BN and the SS-7BN (pink bars)
have a significantly lower postischemic infarct size following
global ischemia compared to
the hypoxic male SS (dark grey
bar).
7. To get the results for the
normoxic male and female
SSBN consomics and the
hypoxic female consomics,
follow these instructions for the
other sub-groups.
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PhysGen hands-on exercises
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Question 4:
1. Go to PhysGen home page (http://pga.mcw.edu), click on link to “Phenotype” under
“Consomic” data. Select Renal B protocol (for mean arterial pressure, MAP phenotype),
click on “Visualization & Statistics” button.
2. Select SS BN consomics since the SS strain develops salt-sensitive hypertension.
Normoxic (atmospheric condition) and high salt diet are the only options in this protocol.
Choose “both” genders, highlight MAP (mmHg) under “select phenotype” and click on
“Do Analysis” button for results.
3. To compare males and females, select “check all strains” button below table, click on
ANOVA analysis button.
4. The ANOVA results indicate that there is a
significant difference but provides no posthoc analysis. To determine whether any of
the consomic strains are significantly
different than the SS strain, select the strain
for comparison (e.g. SS, 21% O2, 8% salt,
male) and click the analysis button on the
right hand side (non-parametric Dunnett
test).
5. These results indicate which strains are
significantly different than the selected
strain (SS parental) by highlighting the
strains in red. The selected strain (SS) is
dark grey and the non-significant strains are
light grey.
6. To get additional information about any of
these strains, a “strain profile” tool is
available. From the main page, select the
“strain profile” under the Consomic Data. A
strain must be selected for comparison to the
parental strain across all protocols.
Question 5:
1. Go to PhysGen home page (http://pga.mcw.edu), click on link to “Phenotype” under
“Consomic” data. Select Lung protocol (for RV/LV weight ratio, right ventricular
hypertrophy phenotype), click on “Visualization & Statistics” button.
2. User must make several choices on this page to get phenotype data results. For this
example, view all three groupings of rats (parental, SS BN consomic, FH BN consomic)
one at a time. First, choose parental strains, choose hypoxic atmospheric conditions.
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PhysGen hands-on exercises
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“Gender” should be both in this case. Scroll down to highlight “rt ventricle/left ventricle
weight ratio” phenotype. Finally,
click “Do Analysis” button for
results.
3. This graph includes all strains
selected (parental strains in this
case), male and female, but only
hypoxic groups. The results are
plotted from lowest to highest
response. To compare males &
females quickly, use the “sort”
button (located to the top left of the
graph) while “gender” is highlighted.
Click “go” to get results.
4. The resulting graph plots the female result next to the male result from the same strain
beginning with the male strain with the lowest response. From this graph, you can see
that some strains have little gender difference (SD, columns 11-12), while other strains
have large differences between genders (WKY, columns 21-22).
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