1 SUPPLEMENTARY INFORMATION (Girirajan et al) Phenotypic evaluation of Rai1 transgenic mice Behavioral testing (modified SHIRPA) Behavioral testing was conducted for 24 mice from line 760 and 11 mice from line 775 along with 51 and 16 non-transgenic littermates, respectively. The tests were performed in the following order by a single observer: physical assessment and simple reflexes, general behavior-observational assessment, sensorimotor reflexes and strength, social interactions and home cage behaviors, and body measurements. Body weight and growth assessment Initially, the mouse was weighed and body measurements, including distance between the bony landmarks of the craniofacial region and the limbs, were taken. In a fume hood, ~300 µl of isofluorane was used to anesthetize the mouse by inhalation. The following measurements (mm) of the mouse were made: outer eye to outer eye, outer ear to outer ear (biparietal diameter), tip of nose to top of head (snout length), length of trunk from mandible, front limbs (toe to knee and knee to hip), hind limbs (toe to knee and knee to thigh). All measurements of transgenic animals were compared to age and sex-matched non-transgenic littermates. Craniofacial and limb measurements In order to assess any craniofacial defects in the Rai1 overexpressing transgenics, bony landmarks of the head were measured. The transgenic mice had close-set eyes (shorter eye- eye distance) with significant effect of genotype (P = 0.008) and age (P < 0.0001), and a shorter ear-ear distance (biparietal diameter) with significant effects of genotype (P > 0.05)
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SUPPLEMENTARY INFORMATION (Girirajan et al) Phenotypic ... · Behavioral testing (modified SHIRPA) Behavioral testing was conducted for 24 mice from line 760 and 11 mice from line
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SUPPLEMENTARY INFORMATION (Girirajan et al)
Phenotypic evaluation of Rai1 transgenic mice
Behavioral testing (modified SHIRPA)
Behavioral testing was conducted for 24 mice from line 760 and 11 mice from line 775 along
with 51 and 16 non-transgenic littermates, respectively. The tests were performed in the
following order by a single observer: physical assessment and simple reflexes, general
behavior-observational assessment, sensorimotor reflexes and strength, social interactions
and home cage behaviors, and body measurements.
Body weight and growth assessment
Initially, the mouse was weighed and body measurements, including distance between the
bony landmarks of the craniofacial region and the limbs, were taken. In a fume hood, ~300
µl of isofluorane was used to anesthetize the mouse by inhalation. The following
measurements (mm) of the mouse were made: outer eye to outer eye, outer ear to outer ear
(biparietal diameter), tip of nose to top of head (snout length), length of trunk from mandible,
front limbs (toe to knee and knee to hip), hind limbs (toe to knee and knee to thigh). All
measurements of transgenic animals were compared to age and sex-matched non-transgenic
littermates.
Craniofacial and limb measurements
In order to assess any craniofacial defects in the Rai1 overexpressing transgenics, bony
landmarks of the head were measured. The transgenic mice had close-set eyes (shorter eye-
eye distance) with significant effect of genotype (P = 0.008) and age (P < 0.0001), and a
shorter ear-ear distance (biparietal diameter) with significant effects of genotype (P > 0.05)
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and age (P < 0.0001) compared to the controls (Supplementary Fig. S3A, B). Rai1 transgenic
mice also have shorter snouts (measured from the top of the head to tip of nose) compared to
the non-transgenic controls with the two-way ANOVA showing genotype (P <0.0001) and
age (P <0.001) (Supplementary Fig. S3C). Limb sizes of the transgenic mice were shorter
(Supplementary Fig. S3D-G); however, when normalized for body lengths, transgenic mice
had no craniofacial or skeletal deformity but had proportionately smaller head and body sizes
when compared to non-transgenic animals.
General behavioral-observational measurement:
Gross motor functions and general behavior were qualitatively assessed as described
previously.1,2 Briefly, the mouse to be tested was placed in a clean empty cage and observed
for 30 min. The presence of a particular behavior during the observation period was given a
positive score. Observed behaviors included wild-running, freezing, sniffing, rearing,
licking, defecation, urination, grooming, gait, and stereotypies.
Physical assessment and simple reflexes:
Physical features were recorded including whisker appearance and length, skin or fur
abnormalities, signs of self-abuse such as bald patches or ulcerated lesions, condition of the
genital/rectal areas, and condition of nails and teeth. Evaluations to elicit simple reflexes:
Righting reflex: the mouse was turned onto its back, and the time to right itself was noted.
Sound orientation: a brief sharp sound was made to the left and right side of the mouse and
the response, such as turning to the direction of the sound, flinching, startle response, or
freezing was noted. Whisker response was measured by touching the whiskers of a freely
moving mouse with a cotton swab. A normal mouse will stop moving its whiskers and will
turn its head to the side of the whiskers that were touched. Eye blink and ear twitch response
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was elicited by touching the corner of the eye and the tip of the ear (pinna), respectively, with
the tip of a cotton swab. Pupil constriction/dilation: constriction of pupil was noted when a
pen-light was shone into the mouse’s eye, and pupil dilation was observed when the light was
removed from the eye.
Cage-top hang test:
Neuromuscular abnormalities were detected by measuring motor strength and coordination
using standard protocols.1,2 The mouse was allowed to hold on to a cage-top which was then
inverted over the cage. The time in seconds (s) the mouse remained hanging was noted. The
average of three trials for each mouse was reported. A 60-s cut-off time was set for a
standard test session.
Sensorimotor reflexes and strength:
Postural reflexes: The mouse was placed in an empty cage and the cage was shaken from
side to side and up and down for 10 s each. The ability of the mouse to maintain an upright
position was noted. Response to being picked up by tail: The normal response of a mouse is
to raise the head, extend its limbs, and reach for the ground when lowered. The presence or
absence of this response was noted. Gait test: The bottom of each foot of the mouse was
coated with non-toxic paint, and the mouse was allowed to walk through a small tunnel on
white paper. Stride length (distance between two rear paw prints), sway length or stride
width, and stance length was measured as described previously.3,4 Footprint measurements of
transgenic and non-transgenic mice were then compared using unpaired Student t test with
post hoc Bonferroni’s corrections. Hot plate test: Mouse was placed on a hot plate set at 55°
C, and the time (s) for the mouse to display a common response (jump, raise paw, paw lick,
or paw shake) or any abnormal response was noted. The maximum trial time was 20 s.
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Social interactions and home cage behaviors:
Mice were housed in 2-5 per cage based on their sex, and were periodically observed for
aggressive behaviors such as barbering (hair loss due to excessive grooming or fur and
whisker trimming). Breeding cages were observed for mutilation of newborn pups.
Nest building and tube tests were performed in order to assess social behaviors as described
previously.1,2 Nest building assessment: Nesting material, a 5 X 5 cm2 piece of cotton was
introduced in a clean cage prior to the behavioral testing. At various time points, 30, 60, 90,
and 120 min, the position of the mouse compared to the nest (mouse huddled on nest, mouse
huddled off nest, nest chewed and fluffy, nest chewed and flat, nest partially chewed and flat,
and nest partially chewed and fluffy), and the quality of the nest was recorded. Tube test: A
transgenic and a non-transgenic mouse of the same gender were placed at opposite ends of a
tube (30 cm in length and 3 cm in diameter) and monitored for dominance (which mouse
pushes the other mouse out of the tube) and submission. The test was repeated with mice
from different cages. Statistical analysis was performed using Chi-square one-sample test to
determine if the number of wins was significantly different than by chance.2,5
Functional observational battery (F.O.B)
At the beginning of F.O.B testing, mice were placed in the open field and scored on the
following measures for the first 5 min of the test: posture, gait, gait abnormalities, arousal,
palpebral closure (all are rank-order data), rearing (number of times the forelimbs are raised
off the surface), and descriptive information on clonic (alternate contractions and relaxation
of muscles) and tonic movements (continuous muscular contractions). Open field
measurements were conducted on a 40 X 76-cm lab cart circumscribed by a 7.5 cm rim.
Subsequently, tests for stimulus reactivity such as approach response (reaction when
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approached with a blunt object), click response (reactivity to an auditory stimulus), touch
response (sides were gently touched with a blunt object), and tail pinch response (metal
tweezers were used to gently squeeze the tail approximately 2 cm from the tip and reaction
elicited) were evaluated over the next 5 min. Tests for psychomotor coordination including
forelimb grip strength, inverted screen task, and hind limb splay were also evaluated.
Forelimb grip strength (measured in kg of force) was determined using a strain gauge (DFG-
2 Chatillon, Greensboro, NC). The forepaws of the mouse were placed on a horizontal bar
mounted on a gauge, and the tail was slowly pulled back. The kg of tension was recorded
from the gauge at the time the mouse released its forepaws from the horizontal bar. The
maximum tension from the five trials was used in the analysis. The inverted screen test
consisted of three practice trials prior to the actual test. Each mouse was placed on a 13 X 13
cm screen, which was then inverted. The time taken (s) for the mouse to climb to top of the
screen, was noted. In the hind limb splay test, the hind limb footpads of each mouse was
smeared with nontoxic paint, and the mouse was dropped from a height of 15 cm onto a paper
surface that was placed over a foam landing pad. Each mouse was tested twice, and the
distance (mm) between the footprints was measured and the mean determined. Locomotor
activity was measured in standard activity chambers (20 X 20 X 30 cm) interfaced with a PC-
operated automated activity monitor. Mice were placed in individual activity chambers, and
spontaneous activity was measured. Using a 16 × 16 Photobeam system (San Diego
Instruments, San Diego, CA), total locomotor counts and central and peripheral counts were
recorded in two 5-min sessions (at age ~20-24 w). The locomotor activity from the center of
the arena was divided by the total locomotor activity to obtain a center/total counts ratio.
Differences in the ratio of the center to total locomotor activity can be used as an indicator of
anxiety, as highly anxious mice tend to avoid the center of an open field.1,6-8 Subsequently,
the mice were evaluated for ease of removal and handling reactivity.
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The tail flick test was used to measure sensitivity of the mouse to a painful stimulus. Tail
flick latency was assessed by holding the mouse gently and placing its tail in the groove of
the tail flick apparatus. The 8V (6 amp) high intensity light (intensity = 13 in range of 1-25)
was illuminated to generate heat, and latency (s) to move the tail away from the light source
was measured. The light automatically extinguished once the tail was removed (or after 10
s).
Circadian activity
Wheel running activity of animals was analyzed in two phases. Mice were first maintained
under light:dark conditions (12L:12D) for one week to measure their ability to entrain to the
lights-off stimulus and to determine their baseline activity across the light:dark cycle. Mice
were then switched to constant dark (DD) conditions for three weeks to establish their
circadian period.9 Data are automatically collected and stored from microswitch closures
onto an IBM computer based acquisition system (developed by Ralph Mistlberger, Simon
Fraser University, British Columbia, Canada). Clocklab, an IBM based interactive program
was used to visualize, analyze and quantify the activity of the animals. The free-running
period (τDD) for each animal was calculated using days 10-20 in DD. These data were
analyzed using an IBM based interface program, WebCircadia. Results are provided in
Supplementary Data (Fig. S4).
Histological and serum analyses
For paraffin histology, whole mouse organs (brain, liver, spleen, heart, kidney, and thymus)
isolated from age and sex-matched transgenic and non-transgenic controls were fixed in 4%
para-formaldehyde in phosphate-buffered saline. Coronal sections were dehydrated in graded
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alcohol series and embedded in paraffin. Sections (3 µM) were mounted on silanized glass
slides and stained with hematoxylin and eosin using standard protocols. Images are provided
in Supplementary data (Fig. S5).
Blood samples were collected from fasted 20 w old mice from each genotype via cardiac
puncture immediately following CO2-induced unconsciousness (n=3 for each genotype).
Serum was separated using Serum-Gel clotting activator micro tubes (Sarsted, Newton, NC).
Samples were analyzed at the Comparative Pathology Laboratory, UC Davis for total
cholesterol, glucose, and levels of liver enzymes (alanine aminotransferase (ALT) and
aspartate aminotransferase (AST).
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Rai1 transgenic mouse expression data
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Fig. S1. Rai1 mRNA and protein expression analysis. A. Real-time expression analysis
showing relative levels of Rai1 cDNA in the Rai1 homozygous transgenic brain (n=2)
compared to wild type (WT). Each reaction was run in triplicate. B. Western blotting on 20
µg of brain and liver protein lysates from Rai1 transgenic animals probed with anti-Rai1
(upper and middle panels) and anti-Srebp1 (bottom panel) antibodies is shown.
Western blotting: Total proteins were isolated from brain and liver samples of transgenic and
non-transgenic mice using radioimmunoprecipitation analysis (RIPA) buffer (150 mM NaCl,
50 mM Tris-HCl, pH 7.5, 500 µM EDTA, 100 µM EGTA, 1.0% Triton X-100, and 1% sodium
deoxycholate) containing protease inhibitors (Sigma-Aldrich, St. Louis, MO). About 20 µg
of the protein lysates were boiled and run on a NuPAGE™ 3-8% Tris-acetate gel using a
XCell SureLock™ Mini-cell (Invitrogen, Carlsbad, CA), and transferred to 0.45 µm PVDF
membrane (Millipore Corporation, Bedford, MA), using XCell II™ Blot Module (Invitrogen,
Carlsbad, CA). The blot was then blocked in PBST (137 mM NaCl, 2.7 mM KCl, 8 mM
Na2HPO4, 2 mM KH2PO4 and 0.05% Tween-20) containing 10% non-fat dry milk overnight
at 4ºC. The blot was then incubated with rabbit anti-Rai1 (1:2000 dilution) or anti-Srebp1
(1:4000) antibody at 37ºC for 1 h, washed 6X, 5 min each, with PBST containing 2% non-fat
dry milk; incubated with HRP-conjugated goat-anti-rabbit secondary IgG antibody (1:20,000,
Vector labs, Burlingame, CA) for 1 h at 37ºC, washed 6X, 5 min each with PBST; and
detected with Western Lightning™ Chemiluminescence Reagent Plus (PerkinElmer, Boston,
MA).
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Body weight data for the 775 line of Rai1 transgenic mice
Fig. S2. Growth assessment of transgenic and non-transgenic mice. Body weights of
male and female transgenic and non-transgenic mice from the 775 line are shown. Data were
analyzed by a two-way ANOVA for genotype [F (3, 71) = 20.80, P<0.001], age [F (3, 71) =
62.71, P<0.001], and interaction [F (9, 71) = 0.71, P=0.6].
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Fig. S3. Craniofacial and body length assessments of Rai1 transgenic and non-
transgenic mice. Craniofacial parameters evaluated include A. eye-eye distance, B.
biparietal diameter (distance between the ears), and C. snout lengths (distance from the top of
head to tip of nose). Male (bold black line) and female (bold gray line) transgenic mice along
with male (dotted black line) and female (dotted gray line) non-transgenic mice at different
time-points (in weeks) are shown. Each gender and genotype n ≥ 15 mice, were utilized for
the study. Data were analyzed by a two-way ANOVA for genotype, age, and interaction.
p<0.05 is considered significant. D-G. Limb measurements for transgenic (T) (bold black
line) and non-transgenic (NT) mice (dotted black line) are shown. Knee to hip (D and F) and
toe to knee (E and G) measurements for front limb and hind limb, respectively, are shown.