german mouse clinic, martin hrab ě de angelis , director
Post on 06-Jan-2016
20 Views
Preview:
DESCRIPTION
TRANSCRIPT
Mouse Line DUF1220KO(Domain Deletion from PDE4DIP: phosphodiesterase 4D interacting protein (myomegalin))
GERMAN MOUSE CLINIC,Martin Hrabě de Angelis, Director
Collaboration withMajesta O‘Bleness, Jonathon Keeney and James SikelaBiochemistry and Molecular GeneticsUniversity of Colorado Anschutz Medical Campus/USA
Body Composition (qNMR)
female male Linear model Linear model Linear model
control mutant control mutant sex genotype body mass
n=15 n=15 n=15 n=13
mean ± sd mean ± sd mean ± sd mean ± sd p-value p-value p-value
Body mass 20.7 ± 1.5 20.8 ± 1.9 25.8 ± 2.4 24.9 ± 2.7 < 0.001 0.49 NA
Fat mass 4.6 ± 0.6 4.7 ± 0.6 5.1 ± 0.6 5.2 ± 0.9 < 0.001 0.035 < 0.001
Lean mass 12.9 ± 0.8 13 ± 1.2 17 ± 1.6 16.1 ± 1.7 < 0.001 0.121 < 0.001
female male Linear model Linear model Linear model
control mutant control mutant sex genotype body mass
n=15 n=15 n=12 n=13
mean ± sd mean ± sd mean ± sd mean ± sd p-value p-value p-value
Body mass 23.8 ± 2.4 23.1 ± 2.1 29.1 ± 2.6 28.5 ± 4 < 0.001 0.386 NA
Fat mass 5.9 ± 1.2 5.3 ± 0.8 6.5 ± 1.1 6.7 ± 1.8 < 0.001 0.629 < 0.001
Lean mass 14.5 ± 1.1 14.5 ± 1.2 18.7 ± 1.6 18.2 ± 2.3 < 0.001 0.519 < 0.001
Body Composition (qNMR)
Linear mixed effects model
sex genotype measurement body mass genotype:measurement
p-value p-value p-value p-value p-value
Body mass
< 0.001 0.467 < 0.001 NA 0.388
Fat mass < 0.001 0.205 0.555 < 0.001 0.055
Lean mass
< 0.001 0.683 0.161 < 0.001 0.005
Body Composition (qNMR) – Only Young
Energy Metabolism: Results
Indirect calorimetry
Female male Linear model Linear model Linear model Linear model
control mutant control mutant sex genotype body mass sex:genotype
n=15 n=15 n=15 n=13
mean ± sd mean ± sd mean ± sd mean ± sd p-value p-value p-value p-value
avg. mass [g]
21 ± 1.5 21.1 ± 1.8 26.1 ± 2.4 25.2 ± 2.7 < 0.001 0.52 NA 0.41
Food intake [g]
2.4 ± 0.4 2.6 ± 0.4 2.7 ± 0.7 2.5 ± 0.6 0.427 0.994 0.677 0.106
avg. VO2 [ml/(h animal)]
81.77 ± 7.44 84.61 ± 8.78 87.01 ± 7.51 84.95 ± 6.45 0.004 0.465 < 0.001 0.378
min. VO2 [ml/(h animal)]
56.27 ± 5.60 57.13 ± 5.01 58.8 ± 7.61 55 ± 6.81 < 0.001 0.544 < 0.001 0.27
max. VO2 [ml/(h animal)]
110.4 ± 10.55 113.6 ± 11.95 121.53 ± 9.91 119.23 ± 11.45 0.075 0.485 < 0.001 0.586
avg. RER [VCO2/VO2]
0.89 ± 0.03 0.89 ± 0.03 0.90 ± 0.04 0.90 ± 0.04 0.42 0.884 NA 0.576
avg. distance [cm] 4909 ± 1576 6236 ± 1459 4585 ± 1769 5589 ± 1802 0.269 0.01 NA 0.711
avg. rearing [counts] 127 ± 92 194 ± 130 98 ± 51 157 ± 122 0.237 0.024 NA 0.883
Energy Metabolism: Results
Energy Metabolism: Conclusions
Duf1220 ko mice were hyperactive both regarding locomotor activity as well as rearing behavior. The hyperactivity had no major effects on energy metabolism related parameters.
Results IpGTT
Fasting induced body mass loss vs. Initial body weight
DUF1220ko mutant mice
Body mass loss due to overnight food withdrawal mainly dependedon initial body mass.No clear genotype effect was detected for body mass loss.
DUF1220ko Estimate CI p-value
(Intercept) -0.3 [ -1.52 , 0.91 ] 0.62
genotypemutant 0.18 [ -0.19 , 0.55 ] 0.33
sexm -0.36 [ -0.82 , 0.09 ] 0.11
bw_before 0.12 [ 0.06 , 0.17 ] <0.001
genotypemutant:sexm 0.12 [ -0.41 , 0.65 ] 0.65
Results IpGTTPlasma glucose concentration [mmol/l] during IpGTT
AUC 0-30 AUC 30-120
We see a trend towards higher AUC values in male mutants, but the differences are quite small with largely overlapping ranges. Therefore this result might be accidental.
DUF1220ko female male Linear model Linear model Linear model
control mutant control mutant genotype sex genotype:sex
n=15 n=14 n=15 n=13
mean ± sd mean ± sd mean ± sd mean ± sd p-value p-value p-value
Glucose (T=0) 4.82 ± 0.71 4.53 ± 0.55 5.15 ± 0.71 5.35 ± 0.77 0.804 0.003 0.19
AUC 0-30 274.04 ± 37.57 290.16 ± 51.13 280.51 ± 66.73 325.07 ± 55.39 0.038 0.152 0.323
AUC 30-120 435.58 ± 88.13 463.38 ± 99.17 568.93 ± 109.89 693.61 ± 177.25 0.022 < 0.001 0.139
Clinical Chemistry Results, Fasting
Means, standard deviation and p-values calculated by a linear model(parameter ~ genotype * sex)
DUF1220ko female maleLinear model
Linear model
Linear model
control mutant control mutant genotype sexgenotype:
sex
n=15 n=15 n=15 n=14
mean ± sd mean ± sd mean ± sd mean ± sd p-value p-value p-value
fasting Glucose [mmol/l] 10.95 ± 1.59a 13.05 ± 2.92a 14.9 ± 3.62 12.48 ± 2.28 0.832 0.026 0.003
fasting Cholesterol [mmol/l] 2.447 ± 0.224a 2.324 ± 0.289a 2.689 ± 0.311 2.738 ± 0.205 0.604 < 0.001 0.231
fasting HDL-cholesterol [mmol/l] 1.824 ± 0.173a 1.735 ± 0.212a 2.031 ± 0.277 2.143 ± 0.15 0.845 < 0.001 0.084
fasting non-HDL Cholesterol [mmol/l]
0.623 ± 0.071a 0.589 ± 0.089a 0.657 ± 0.157 0.595 ± 0.1 0.116 0.505 0.629
fasting Triglycerides [mmol/l] 0.716 ± 0.134a 0.704 ± 0.166a 0.784 ± 0.107 0.753 ± 0.154 0.57 0.131 0.794
fasting NEFA [mmol/l] 0.58 ± 0.14a 0.6 ± 0.23a 0.58 ± 0.15 0.67 ± 0.16 0.236 0.495 0.423
fasting Glycerol [mmol/l] 0.192 ± 0.048a 0.162 ± 0.049a 0.153 ± 0.03 0.165 ± 0.055 0.471 0.157 0.088
Four female animals – 1 control, 3 mutant – not tested because of too low body weight.Glucose
Clinical Chemistry Results
Means, standard deviation and p-values calculated by a linear model(parameter ~ genotype * sex)
Sodium
One female Mut excluded ID 30264548 > severe kidney disease
Chloride
DUF1220ko female maleLinear model
Linear model
Linear model
control mutant control mutant genotype sexgenotype:
sex
n=15 n=14 n=12 n=13
mean ± sd mean ± sd mean ± sd mean ± sd p-value p-value p-value
Sodium [mmol/l] 143 ± 1 143 ± 1 147 ± 1 145 ± 2 0.031 < 0.001 0.032
Potassium [mmol/l] 3.8 ± 0.4 3.8 ± 0.3 4.3 ± 0.2 4.1 ± 0.2 0.059 < 0.001 0.273
Chloride [mmol/l] 106.8 ± 1.3 106.9 ± 2.1 108.5 ± 1.2 106.9 ± 1.7 0.104 0.067 0.066
Total protein [g/l] 52.7 ± 2.8 52.8 ± 2 53.9 ± 1.1 53.8 ± 1.9 0.989 0.057 0.857
Albumin [g/l] 28.3 ± 1.5 28.3 ± 1.3 27.5 ± 0.9 27 ± 1.3 0.399 0.003 0.519
Creatinine enz. [mumol/l] 14.73 ± 0.98 15.2 ± 1.61 15.12 ± 2.21 15.25 ± 1.42 0.496 0.618 0.699
Urea [mmol/l] 10.58 ± 1.72 10.14 ± 0.96 11.81 ± 1.34 11.76 ± 1.27 0.508 < 0.001 0.606
Potassium
Clinical Chemistry: Results
Means, standard deviation and p-values calculated by a linear model (parameter ~ genotype * sex)
ASATALAT LDH
DUF1220ko female maleLinear model
Linear model
Linear model
control mutant control mutant genotype sexgenotype:
sex
n=15 n=14 n=12 n=13
mean ± sd mean ± sd mean ± sd mean ± sd p-value p-value p-value
Cholesterol [mmol/l] 2.024 ± 0.265 2.024 ± 0.284 2.343 ± 0.237 2.288 ± 0.188 0.684 < 0.001 0.688
Triglyceride [mmol/l] 0.975 ± 0.185 0.888 ± 0.205 1.255 ± 0.305 1.255 ± 0.434 0.588 < 0.001 0.587
ALAT/GPT [U/l] 25 ± 4 27 ± 5 36 ± 15 66 ± 57 0.04 0.003 0.084
ASAT/GOT [U/l] 53 ± 14 57 ± 15 56 ± 14 87 ± 71 0.081 0.097 0.177
alpha-Amylase [U/l] 572.42 ± 59.29 577.08 ± 61.36 776.37 ± 153.99 790.11 ± 171.33 0.779 < 0.001 0.89
Glucose [mmol/l] 13.3 ± 1.4 13.32 ± 1.53 13.18 ± 2.08 13.66 ± 1.43 0.576 0.808 0.605
LDH [U/l] 125.7 ± 38.3 137.9 ± 33.9 172.5 ± 56.7 255 ± 168.1 0.061 0.002 0.161One female Mut excluded ID 30264548 > severe kidney disease
Clinical Chemistry: Results
Means, standard deviation and p-values calculated by a linear model (parameter ~ genotype * sex)
ALPOne female Mut excluded ID 30264548 > severe kidney disease
DUF1220ko female maleLinear model
Linear model
Linear model
control mutant control mutant genotype sexgenotype:
sex
n=15 n=14 n=12 n=13
mean ± sd mean ± sd mean ± sd mean ± sd p-value p-value p-value
Calcium [mmol/l] 2.45 ± 0.08 2.43 ± 0.08 2.34 ± 0.1 2.34 ± 0.08 0.75 < 0.001 0.728
Inorganic phosphate [mmol/l] 1.6 ± 0.2 1.6 ± 0.3 1.6 ± 0.3 1.6 ± 0.3 0.55 0.857 0.99
Iron [mumol/l] 24.9 ± 4.3 23.8 ± 4.1 19.6 ± 2.9 20.7 ± 2.4 0.986 < 0.001 0.26
ALP [U/l] 164 ± 26 176 ± 22 106 ± 15 95 ± 15 0.91 < 0.001 0.046
Means, standard deviation and p-values calculated by a linear model (parameter ~ genotype * sex)
Hematology: Results
DUF1220ko female male Linear model Linear model Linear model
control mutant control mutant genotype sex genotype:sex
n=15 n=15 n=12 n=12
mean ± sd mean ± sd mean ± sd mean ± sd p-value p-value p-value
RBC [Mio/mm³] 11.16 ± 0.58 11.29 ± 0.58 11.19 ± 0.65 11.08 ± 0.61 0.972 0.592 0.459
HGB [g/dl] 17.27 ± 0.76 17.53 ± 0.87 16.57 ± 1.12 16.48 ± 1.17 0.752 0.002 0.531
HCT [percent] 53.79 ± 2.63 54.49 ± 2.84 54.73 ± 2.53 54.95 ± 2.18 0.516 0.331 0.738
MCV [fl] 48.33 ± 0.9 48.4 ± 0.74 49 ± 2.09 49.75 ± 1.29 0.259 0.007 0.344
MCH [pg] 15.49 ± 0.55 15.53 ± 0.37 14.79 ± 0.54 14.87 ± 0.49 0.688 < 0.001 0.877
MCHC [g/dl] 32.13 ± 0.83 32.16 ± 0.66 30.3 ± 1.64 29.98 ± 1.48 0.662 < 0.001 0.59
RDW [percent] 14.89 ± 0.41 14.81 ± 0.46 15.24 ± 0.37 15.25 ± 0.72 0.794 0.005 0.748
WBC [10³/mm³] 12.23 ± 3.04 11.49 ± 3.27 11.32 ± 2.69 11.08 ± 3.48 0.573 0.449 0.776
PLT [10³/mm³] 967.2 ± 95.54 948.8 ± 123.16 1246.75 ± 191.89 1262.67 ± 253.32 0.979 < 0.001 0.715
MPV [fl] 6.12 ± 0.15 6.13 ± 0.1 6 ± 0.17 6 ± 0.18 0.936 0.004 0.936
PDW [fL] 5.37 ± 0.24 5.37 ± 0.16 5.25 ± 0.18 5.19 ± 0.24 0.609 0.013 0.609
PLCR [percent] 1.78 ± 0.39 2 ± 0.33 1.59 ± 0.61 1.66 ± 0.53 0.266 0.043 0.55
PCT [percent] 0.59 ± 0.06 0.58 ± 0.07 0.75 ± 0.11 0.75 ± 0.15 0.929 < 0.001 0.857
Data of one animal excluded due to technical problems (30264526, male mutant)
No evidence of remarkable genotype-related differences.
Behaviour: OF Results
Forward locomotion
No significant genotype effects on locomotor activity
Behaviour: OF Results
Vertical locomotion
No genotype effect on rearing activity
Behaviour: OF Results
Anxiety-related behaviour
No genotype effects on centre time
Behaviour: Acoustic Startle Reactivity
Increased ASR by the female mutant mice
Behaviour: Prepulse Inhibition
No genotype effect on PPI
Neurology: SHIRPA-Results
No difference in SHIRPA parameters
Locomotor activity measured as the numbers of squares crossed in the first 30 seconds after transfer into the vieving arena.
No difference in grip strength
Neurology: Results
Since we are especially interested in mitochondria function we measure as a marker lactate levels in collaboration with the Clinical Chemistry Screen
Neurology: Results
White 2001, Sex Transm Infect 2001;77:158-173 doi:10.1136/sti.77.3.158
No difference
No difference in mean latencies, usual improvement over trials,But slightly more often falling
Neurology: Results
Hotplate Test
Nociception: Results
Type of reaction on hotplate
mean ± sd mean ± sd mean ± sd mean ± sd pairwise(Tukey)
pairwise(Tukey)
ANOVA ANOVA ANOVA
female female male male females malescontrol mutant control mutant mutant-
controlmutant-control
genotype sex sex:genotype
N=15 N=15 N=15 N=13 adj. p-value adj. p-value p-value p-value p-valueFirst response time
8.2 ± 2.65 9.26 ± 2.94 10.98 ± 3.48 8.78 ± 2.85 0.768 0.224 0.52 0.125 0.043
Second response time
20.84 ± 33.6 13.07 ± 2.77 15.64 ± 4.79 13.52 ± 3.89 0.616 0.988 0.275 0.618 0.541
Female Male
Control Mutant Control Mutant
n=15 n=15 n=15 n=13
1st response type
hind paw licking 4 8 9 4
hind paw shaking 11 7 6 9
2nd response type
hind paw licking 11 7 6 9
hind paw shaking 4 8 9 4
0 = no reaction at all
1 = no Preyer reflex
2 = retarded reaction
3 = normal reaction
4 = strong reaction
5 = particularly strong reaction
Dysmorphology: Results
Clickbox Test
Score Males Females
Control Mutant Control Mutant
0 7 4 12 13
1 - - - -
2 4 4 2 -
3 4 5 1 2
4 - - - -
5 - - - -
Median 2.00 2.00 0.00 0.00
BL6 background→ become deaf with age
→ no significance (Fisher’s Exact test)
Cardiovascular: Results Echocardiography
Cardiovascular: Results Electrocardiography
Cardiovascular: Results Electrocardiography
** *
Eye: Results
Scheimpflug Imaging
control Duf1220
Eye: Results
Scheimpflug Imaging
Eye: Results
Virtual Drum (vision test)
Levels of Immunoglobulins in Blood Plasma
Levels of Immunoglobulins in Blood Plasma [µg/ml]
Frequencies of Leukocyte Subsets in Blood [% of respective parent gate]
Frequencies of Leukocyte Subsets in Blood [% of all leukocytes]
Lower frequencies of T cells and a slightly lower CD4: CD8 T cell ratio in mutants of both sexes
IgE
female male
female male overallcontrol mutant control mutant
13 14 9 7median median median median
p-value p-value p-value[25%, 75%] [25%, 75%] [25%, 75%] [25%, 75%]
88.7 [57.3,114.8]
90.2 [46.5,182.4]
36.4 [16.7,46.7]
75.4 [32.95,81.35]
0.896 0.365 0.643
IgE Results
•Total plasma IgE in Duf1220KO mice (1st sample)Median, first and third quartile and p-values calculated by a Wilcoxon rank-sum test.
IgE Results
Box plot of total plasma IgE in Duf1220KO mice ng/mlNo statistically significant differences in the IgE levels between control and mutants mice
TEWL Measurement
female male female male overall
control mutant control mutant
n = 15 n = 15 n = 15 n = 13
[25%, 75%] [25%, 75%] [25%, 75%] [25%, 75%] p-value p-value p-value
28.1 [26.65,33.4]
30.3 [29.05,33.15]
33.2 [29.75,39.4]
36.1 [27.1,46.2]
0.34 0.892 0.856
Box plot of transepidermal water loss (TEWL) [g/(m²h)] in Duf1220KO miceMedian, first and third quartile and p-values calculated by a Wilcoxon rank-sum test.
NO statistically significant differences in the TEWL levels between control and mutants male mice.
TEWL Measurement
Lung Function Parameters: Duf1220KO Data are presented as mean SD.
Parameter Female Controln=6
Female Mutant n=5
Wilcoxon-test
Body Mass (BM) [g] 24 ± 3.8 23.9 ± 3.9 1
Volumetric Parameters
Tidal Volume (TV) [ml] 0.205 ± 0.009 0.209 ± 0.004 0.424
Inspiratory Capacity (IC) [ml] 0.79 ± 0.15 0.916 ± 0.059 0.082
Expiratory Reserve Volume (ERV) [ml] 0.16 ± 0.05 0.16 ± 0.04 0.894
Vital Capacity (VC) [ml] 0.96 ± 0.19 1.06 ± 0.06 0.128
Functional Residual Capacity (FRC) [ml] ND ND
Residual Volume (RV) [ml] ND ND
Total Lung Capacity (TLC) [ml] ND ND
Forced Vital Capacity (FVC) [ml] 0.973 ± 0.176 1.079 ± 0.059 0.082
Flow Parameters
Forced Expiratory Volume (FEV100) [ml] 0.944 ± 0.156 1.037 ± 0.05 0.082
Peak Expiratory Flow (PEF) [ml/sec] 30.7 ± 2.1 31.6 ± 3.9 0.266
Mechanical Parameters
Static Lung Compliance (Cchord) [ml/cm H2O] 0.0528 ± 0.0121 0.0569 ± 0.013 0.429
Dynamic Lung Compliance (Cdyn.) [ml/cm H2O] 0.0239 ± 0.005 0.025 ± 0.0024 0.452
Lung Resistance (R) [cm H2O/ml/sec] 1.4 ± 0.11 1.34 ± 0.08 0.329
Lung Function: Results
Pathology: Results Organ Weight
female male female male overall
control mutant control mutant
n=9 n=9 n=8 n=8
median [25%, 75%]
median [25%, 75%]
median [25%, 75%]
median [25%, 75%]
p-value
p-value
p-value
Heart weight [mg] 121
[119 , 128]
126 [117 , 126]
155.5 [148.5 , 162.8]
143.5 [136.8 ,
149] 0.983 0.13 0.448
Tibia length [mm] 17.9
[17.8 , 18.2]
17.9 [17.6 , 18]
18.1 [17.9 , 18.3]
18.3 [18.1 , 18.4]
0.474 0.442 0.899
Liver weight [g] 1.182
[1.109 , 1.209]
1.135 [1.112 , 1.255]
1.685 [1.59 , 1.714]
1.618 [1.56 , 1.772]
0.863 1 0.939
Spleen weight [g] 0.095
[0.088 , 0.116]
0.098 [0.085 , 0.121]
0.084 [0.079 , 0.088]
0.087 [0.084 , 0.098]
0.65 0.456 0.438
Body weight [g] 23.176 [22.11 , 23.714]
22.43 [20.958 , 23.882]
32.517 [29.934 , 32.773]
29.596 [28.241 , 33.197]
0.605 0.505 0.474
Heart Weight / Tibia Length [mg/mm]
6.8 [6.51 , 7.36]
7 [6.64 , 7.16]
8.64 [8.19 , 8.96]
7.82 [7.54 , 8.07]
0.931 0.052 0.362
Heart Weight / Body Weight [mg/g]
5.4 [5.07 , 5.78]
5.59 [5.37 , 5.62]
4.89 [4.62 , 5.17]
4.92 [4.42 , 5.03]
0.589 0.798 0.925
Liver Weight / Tibia Length [mg/mm]
65.85 [62.94 , 66.43]
64.34 [61.78 , 69.99]
91.12 [88.08 , 92.81]
88.22 [85.81 , 94.39]
1 0.645 0.708
Liver Weight / Body Weight [mg/g]
50.88 [47.58 , 52.17]
52.89 [50.86 , 53.72]
52.72 [51.56 , 53.72]
53.09 [52.52 , 54.17]
0.113 0.328 0.073
Spleen Weight / Tibia Length [mg/mm]
5.27 [4.95 , 6.44]
5.71 [4.72 , 6.66]
4.67 [4.34 , 4.91]
4.8 [4.59 , 5.12]
0.649 0.524 0.546
Spleen Weight / Body Weight [mg/g]
4.14 [3.9 , 4.46]
4.11 [3.84 , 4.94]
2.73 [2.51 , 2.93]
2.94 [2.86 , 3.01]
0.605 0.234 0.518
Pathology: Results Organ Weight
No statistically significant difference of total and normalized heart weight was detected comparing mutant and control mice.
Slide 43
Skin NAD Pancreas NAD
Musculoskeletal system NAD Cervical lymph node NAD
Eyes NAD Thymus NAD
Brain NAD Spleen NAD
Cerebellum NAD Thyroid NAD
Heart / Arteries NAD Parathyroid NAD
Trachea NAD Adrenal gland NAD
Lung NAD Kidneys NAD
Teeth NAD Urinary bladder NAD
Salivary gland NAD Testes NAD
Esophagus NAD Epididymis NAD
Stomach NAD Funiculus spermaticus NAD
Small intestine NAD Ovaries NAD
Large intestine NAD Uterus NAD
Liver NAD Vagina NAD
Mamma NAD Body weight NAD
Pathology: Results Overview
top related