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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.

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Fig. S4. Circadian analysis. Voluntary wheel running activity for Rai1 hemizygous

transgenic mice and wild type littermates is shown. Mice were housed individually for 4 w

with free access to the wheel. A. Activity was recorded for female mice and B. male mice,

as the number of wheel turns per minute. Data are plotted as mean activity. The first 12 h

represents the light phase of the cycle. No significant difference was seen between Rai1

transgenics and wild type littermates. C. Analysis of the circadian period is shown in the

histogram of mean free running activity during constant dark condition (τDD ± SEM) for

Rai1 hemizygous transgenics and wild type littermates. A difference of 6 min in the total

circadian rhythm between the transgenic heterozygotes and wild types was noted (P>0.05).

ZT represents the Zeitgeber time, the number of hours elapsed since “lights-on”, where ZT =

0 for light onset and ZT = 12 for dark onset, by convention.

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Fig. S5. Organ analysis. H & E stained histological sections of major organs from

transgenic (T) and non-transgenic (NT) mice are shown. A. Coronal sections of the

ventricular wall (heart) at 20X and whole heart at 1.25X (inset), B. Spleen showing red

(eosin) and white pulp (hematoxylin), C. Sections of the cerebral cortex (brain) at 20X and

10X (inset), D. Sections of kidney showing the outer cortex with glomeruli (arrows) at 20X

and whole kidney at 1.25X (inset), E. Liver showing liver lobules with hepatocytes (arrows)

and central veins, illustrating greater nuclear:cytoplasmic ratio, and F. Thymus.

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Fig. S6. Liver and metabolic tests. Results of tests to analyze the levels of liver enzymes

(A) and metabolites (B) are shown. Comparisons between data from transgenic and non-

transgenic mice were performed using unpaired t test. *P<0.05.

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Fig. S7. Stimulus reactivity tests demonstrating hyperactive behavior in homozygous

transgenic mice. A. The response of mice to handling was graded in the order of increasing

difficulty: easy, low resistance, freezes, squirms, and bites (as shown on the X-axis).

Percentage of mice showing such a response is documented on the Y-axis, n=12 (non-

transgenic), n=11 (hemizygous transgenic), and n=9 (homozygous transgenic). Note that

33% of homozygous transgenic mice bite compared to 11% of hemizygous transgenics and

6% of the non-transgenic mice when handled. B. Ease of removal of mice from the cage was

graded as very easy, easy, moderately difficult, difficult, and very difficult. Approximately

40% of homozygous transgenic mice were very difficult to grab compared to 13% of

hemizygous transgenic and 9% of non-transgenic mice. This is well illustrated by the

supplementary video.

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Supplementary video file. This video depicts the difficulty in removing or handling a Rai1

homozygous transgenic mouse as compared to the non-transgenic mouse. Hyperactive and

possibly anxiety-induced behaviors are also seen.

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Supplementary table 1. Results of modified SHIRPA protocol in Rai1 transgenic mice (775 line)*

Rai1 transgenics (775 line)

Non-transgenics (775 line)

Physical assessment and simple reflexes (% abnormal) Whisker appearance 9 6 Bald patches (% with) 18 0 Nails, teeth, or genitalia 0 0 Righting reflex 0 0 Sound orientation 0 0 Whisker response 9 6 Eye blink/ ear twitch 0 0 Pupil constriction/dilation 0 0

General observational measurement (% with) Wild running1 18 0 Freezing 9 0 Sniffing 100 100 Licking 100 100 Rearing 81 50 Defecation 100 100 Urination 100 100 Stereotypies 0 0

Sensorimotor reflexes and strength (% abnormal) Postural reflex 0 0 Response to being picked up by tail 0 0

Social interactions and home cage behaviors Nest building assessment (% abnormal) 0 0 Tube test for dominance (% of wins) 60 40

*n=11 for transgenics and n=16 for non-transgenics

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