| Basic
Information for Directing a Mouse Atherosclerosis
Project
For an excellent review of the fundamentals
of designing and analyzing mouse atherosclerosis
projects, refer to Daugherty & Rateri
(2005), Methods 36:129-138.
- It is better to set up simultaneous multiple breeding
cages that will give
rise to many, closely coordinated
waves of mice for the final
study rather than to do it
in piecemeal fashion. Make sure the mice are
ear-marked at the time of genotyping.
- When the pups for the study are weaned,
place in cages of
same sex. Each cage should
have a similar number of
mice. Do not enter into the study any mice
that appear abnormal.
- Keep a careful table that identifies
the (1) breeding cage/parents
from which the mouse arose;
(2) genotype; (3) sex; (4) birth date; (5)
two weights: at 6 weeks of age (which the time
special diet usually begins) and at time of
harvest. Assign each mouse an ID# whose code
is only known by the director of the project—George
must be blinded as
to the identity of the mice.
- On a dedicated calendar, note dates in
future for start of special
diet feeding (e.g.,
Western diet if applicable)
and dates of harvest. Make
sure proper diets are received
well before diet-start date. Inform George
and Ira one week before harvest date.
- Make notations on this table of any unusual
events, such as empty
food cages, power shutdowns,
and health issues related
to the mouse room (e.g.,
pinworm outbreak)
or to individual mice (e.g.,
fighting with cage
mates, loss of hair, bleeding,
etc.).
- On late afternoon prior to harvest day:
place mice in fresh,
clean cage and fast overnight.
- On morning of harvest, work with George
to obtain tail clips,
weights, and blood for plasma
cholesterol and lipoproteins.
Label three sets of Eppendorf tubes with the
ID#'s of the mice:
- The first set is for the blood and
should be
pre-filled
with 20 µl of 100mM mM EDTA
in
PBS. These tubes are centrifuged at 14,000
rpm for 10 min in the cold room to isolate
plasma.
- Add this plasma to the second set
of tubes,
which should
be pre-filled
with 10 µl
sodium azide
from a 2%
stock solution;
store the
plasma at 4°C.
- The third set of tubes is for the
tail clips
(in case
genotype
verification is needed)—store
these
at -80°C.
- George will then harvest proximal aorta
and brachiocephalic artery and process by freezing
in OCT or by paraffin
embedding, depending on the procedure. Make
sure that you and George are in sync regarding
the identification of the mice and specimens.
- From the fresh plasma, prepare HDL by
precipitation of apoB-containing
lipoproteins (via kit), and
store the HDL under argon
at 4°C. Within one month,
assay cholesterol and, if
necessary, triglyceride in the plasma (total
cholesterol [TC]) and HDL using the colorimetric
kit. Note that the cholesterol assay is best
in the 2-9 mg/ml range, although HDL is usually
below this. If cholesterol above 12-15 mg/ml,
you will need to dilute with 0.15 M NaCl/1
mM EDTA.
- For FPLC lipoprotein profile: assay a
small aliquot of fresh plasma samples
for total cholesterol and
HDL cholesterol. After retrieving
the data, find mice whose
TC and HDL levels are close to that of the
mean for that sex and genotype based upon your
previous data. For each sex and genotype, pool
the remaining plasma from 2-3 mice that meet
these criteria and fractionate by FPLC. Assay
80 µl
of each fraction for cholesterol.
- A minimum of 300 µl pooled plasma
is
needed. Dilute
with FPLC
buffer (below) if cholesterol is > 15
mg/ml.
- We use Henry Ginsberg's FPLC, run
by Colleen
on P&S 9 th floor cold room.
- Buffer (0.15 M NaCl/1 mM EDTA/0.02%
sodium azide)
must be filtered
to remove
particles using our
sterilizing
1-L filters, degassed in an Erlenmeyer
flask under vacuum, and pre-cooled. Use
Program #2 on FPLC—flow
rate is 0.3
ml/min, and
fractions are ~0.5 ml
last updated: June 14, 2005
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