G2C::Genetics

SAP97 knock-out mouse

S.G.N. Grant and the G2C Consortium

Corresponding email: Seth.Grant@ed.ac.uk  

 

G2CMine Data Warehouse

Dlg1 @ G2CMine

Genetic and Genomic Information

Gene symbol Dlg1
MGI ID MGI:107231
G2Cdb mouse G00000863
Ensembl mouse ENSMUSG00000022770
G2Cdb human G00002112
Ensembl human ENSG00000075711

G2CMine Data Warehouse

G2CMine integrates the scientific findings of the Genes to Cognition Programme that utilised neuroproteomics, psychiatric genetics, high-throughput mouse gene targeting combined with behavioural and electrophysiological phenotyping and informatics in order to develop a general strategy for understanding cognition at the molecular, cellular and systems neuroscience levels.

G2CMine provides comprehensive Gene Ontology, Mammalian Phenotype Ontology, Human Phenotype Ontology, UniProt, genetic and protein interactions, and regional mouse brain expression results, together with the phenotyping results of the G2C Programme.

Mutation

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A mouse embryonic stem (ES) cell line (AJ0497, strain 129/Ola) with an insertional mutation in Dlg1 was obtained from Sanger Institute Gene Trap Resource (SIGTR - sanger.ac.uk/PostGenomics/genetrap/). The insertional mutation in AJ0497, by the gene-trapping vector, pGT0lxr, that was designed to create an in-frame fusion between the 5' exons of the trapped gene and a reporter, β-geo (a fusion of β-galactosidase and neomycin phosphotransferase II), occurred in intron 5-6. Thus, the gene-trapped locus is predicted to yield a fusion transcript containing exons 1–5 of Dlg1 and β-geo.

The ES cells were injected into C57BL/6 blastocysts to create chimeric mice, which were bred with 129S5 mice to generate heterozygous (+/–)Dlg1 mutant mice. Those F1 heterozygous mice had been backcrossed with 129S5 mice for 1-2 times before being used for intercrossing.

Primers used for genotyping (a, b, c and d). PCR genotyping of gene trap SAP97 mice using forward primers, a and c, and reverse primers, b and d, to amplify the wt and mutant alleles respectively.

Genotyping

Genomic DNA was isolated from ES cells or mouse tissues by Wizard SV 96 DNA purification system (Promega Cat A2371). Genotyping PCR consisted of 2 independent reactions. A 421bp product amplified from the wild-type (wt) allele using a forward primer a (5'- CAGTCCTCAAATCCCATCGG -3') upstream of the cassette and a reverse primer b (5'- CACTGTCTGAAGGAACACTG -3') in the wt sequence deleted by targeted mutation. The targeted allele is detected using forward primer c (5’- CTGTCCTGGAACTCACTCTG –3’) with reverse primer d (5’- CTCAAAGTCAGGGTCACAAGG -3’) within the gene trap cassette and amplifies a 500bp product. After enzymatic amplification for 35 cycles (45 seconds at 94 °C, 45 seconds at 55 °C, and 1 minute at 72 °C), the PCR products were size-fractionated on a 2% agarose gel in 1x Tris borate-EDTA buffer (image not shown).

Expression

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Total RNA (100μg) was isolated from ES cells with RNeasy Mini kit (Qiagen, Cat 74104). RT-PCR was performed by generating first strand cDNA using Oligo(dT)12-18 primer (Invitrogen, Cat 18418-012) and superscript II reverse transcriptase (Invitrogen, Cat 18064-071). RT-PCR consisted of a 163bp product amplified from the wild-type (wt) cDNA using a forward primer X (5’- GTCCATGTCTCAGAGAAGAAC -3’) upstream of the cassette and a reverse primer Y (5’- CAGGACGACAGTACTCTCAG -3’) downstream of the insertion site. A 185bp product was amplified from the gene trap cDNA using primer X with reverse Primer Z (5’- GATCCTCTAGAGTCCAGATCTG -3’), within the β-geo cassette. After enzymatic amplification for 35 cycles (45 seconds at 94 °C, 1 minute at 55 °C, and 1 minute at 72 °C), the PCR products were size-fractionated on a 2% agarose gel in 1x Tris borate-EDTA buffer(image not shown).

Western blot to confirm reduced protein expression. Protein expression of Sap97 protein was confirmed using immunoblotting of hippocampal extracts from SAP97 heterozygous mutant mice and wildtype mice. There was significant reduction in Sap97 protein levels in the SAP97 +/- mutant mouse (p<0.04, n=4pairs) compared with wildtype mice.

Breeding

No SAP97-/- mice were produced from SAP97+/- intercrosses. Male and female SAP97+/- mice developed normally to adulthood, were fertile, exhibited normal body size and no gross abnormalities. Genotypes of 3-week-old pups from SAP97+/- intercrosses identified 70 wt and 99 SAP97+/- progeny (Χ2 p= <0.001). Backcrosses onto the 129S5/SvEvBrd background were used to maintain the colony and to generate heterozygous and wildtype mice to study.

Overview

Mutant mice showed no discernible overall behavioural difference from wildtypes. With heterozygous genotype and no behavioural variables significantly affected in mutants, this mutation was deemed haplosufficient.

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The G2CMine data warehouse provides cohort summaries and individual mouse observations from the SAP97 knock-out line phenotyping.

 

Variables shown are: EPM total distance, Total distance (cm) travelled in any arm or central zone of the EPM. EPM max speed, Maximum speed (cm/s) travelled in any arm or central zone of the EPM. EPM % time in open, Percentage of time in the open or closed arms of the EPM spent in open arms. EPM time in centre, Total time (s) spent in the central zone of the EPM. EPM max speed, open vs closed, Difference between the maximum speed (cm/s) observed in the open arms and the closed arms of the EPM. OF, NOE total distance, Total distance travelled (log₁₀ cm) during initial exposure to the open field and in presence of the novel object. NOE vs OF distance travelled, Difference in distance travelled (cm) in presence of the novel object and during initial exposure to open field. RR naive fall time, Fall time on accelerating rotarod (log₁₀ s), naive performance in session 1. RR learning, Learning on rotarod, measured as increase in fall time per trial (s/trial) in session 1. RR memory, Memory on rotarod, measured as excess fall time at middle of session 2 relative to middle of session 1. Fear learning, trial effect, Fear learning, measured as extra % time freezing before third trial compared to % time freezing before first trial. Fear learning, tone effect, Fear learning, measured as increase in % time freezing due to third tone compared to increase in % time freezing due to first tone. Contextual memory, mean, Contextual memory, measured as difference in % time freezing during first 120 s re-exposure to the box compared to first 120 s in the box on previous day. Contextual memory, change, Contextual memory, measured as increase in % time spent freezing from first time bin of 30 s to fourth bin of 30 s during 120 s re-exposure to the box. Cued memory, mean, Cued memory, measured as increase in % time spent freezing during 120 s of tone re-exposure compared to increase in % time spent freezing during initial tone on previous day. Cued memory, change, Cued memory, measured as increase in % time spent freezing from first time bin of 30 s to fourth bin of 30 s during 120 s re-exposure to the tone.

 

Variable Units Wildtype M (n=13) Wildtype F (n=13) Mutant M (n=14) Mutant F (n=21) P(sex×mutation) P(mutation)
EPM total distance cm 719 (76) 790 (48) 716 (64) 810 (53) 0.85 0.88
EPM max speed cm/s 16 (1.1) 16.8 (1) 14.9 (0.9) 17 (0.8) 0.48 0.66
EPM % time in open % 30.9 (8.2) 17.9 (5.3) 13.7 (2.5) 23.3 (5.4) 0.057 0.4
EPM time in centre s 129 (15.9) 140.8 (8.4) 133.7 (17.1) 135.5 (9) 0.7 0.96
EPM max speed, open vs closed cm/s -5.8 (2.3) -6.5 (1.5) -4 (0.9) -4.9 (1.1) 0.94 0.26
OF, NOE total distance log10 cm 3.15 (0.08) 3.31 (0.08) 3.03 (0.05) 3.24 (0.06) 0.69 0.21
NOE vs OF distance travelled cm -255 (156) -49 (145) -179 (99) -26 (95) 0.83 0.7
RR naive fall time log10 s 0.96 (0.11) 0.9 (0.08) 0.77 (0.09) 1.07 (0.07) 0.04 * 0.95
RR learning s/trial 0.3 (0.7) 1.3 (0.5) 1.2 (0.7) 1.8 (0.5) 0.68 0.25
RR memory s 11.1 (7.1) 8.3 (3.4) 4.8 (2.2) 5.4 (3.6) 0.7 0.31
Fear learning, trial effect % freezing 53.6 (7.2) 57.5 (7.3) 51 (4.2) 52.1 (6.2) 0.83 0.53
Fear learning, tone effect % freezing -0.9 (6) -4.8 (5.2) 4.9 (5.2) 1.5 (4.3) 0.97 0.25
Contextual memory, mean % freezing 41.9 (5.6) 42.8 (5.6) 43.3 (5.6) 46.1 (4.3) 0.86 0.65
Contextual memory, change % freezing 30.4 (9.6) 29.4 (7.6) 36.7 (6.5) 23.9 (5.6) 0.42 0.99
Cued memory, mean % freezing -4.6 (7.5) 4.2 (7.2) 0.8 (3) 12.5 (3.9) 0.79 0.21
Cued memory, change % freezing -1.2 (8.7) 0.4 (6.3) -6.6 (4.8) -3.7 (3.3) 0.91 0.41

Elevated Plus Maze

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Variables shown are: EPM total distance, Total distance (cm) travelled in any arm or central zone of the EPM. EPM max speed, Maximum speed (cm/s) travelled in any arm or central zone of the EPM. EPM % time in open, Percentage of time in the open or closed arms of the EPM spent in open arms. EPM time in centre, Total time (s) spent in the central zone of the EPM. EPM max speed, open vs closed, Difference between the maximum speed (cm/s) observed in the open arms and the closed arms of the EPM.

 

Variable Units Wildtype M (n=13) Wildtype F (n=13) Mutant M (n=14) Mutant F (n=21) P(sex×mutation) P(mutation)
EPM total distance cm 719 (76) 790 (48) 716 (64) 810 (53) 0.85 0.88
EPM max speed cm/s 16 (1.1) 16.8 (1) 14.9 (0.9) 17 (0.8) 0.48 0.66
EPM % time in open % 30.9 (8.2) 17.9 (5.3) 13.7 (2.5) 23.3 (5.4) 0.057 0.4
EPM time in centre s 129 (15.9) 140.8 (8.4) 133.7 (17.1) 135.5 (9) 0.7 0.96
EPM max speed, open vs closed cm/s -5.8 (2.3) -6.5 (1.5) -4 (0.9) -4.9 (1.1) 0.94 0.26

Open Field/Novel Object

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Variables shown are: OF, NOE total distance, Total distance travelled (log₁₀ cm) during initial exposure to the open field and in presence of the novel object. NOE vs OF distance travelled, Difference in distance travelled (cm) in presence of the novel object and during initial exposure to open field.

 

Variable Units Wildtype M (n=13) Wildtype F (n=13) Mutant M (n=14) Mutant F (n=21) P(sex×mutation) P(mutation)
OF, NOE total distance log10 cm 3.15 (0.08) 3.31 (0.08) 3.03 (0.05) 3.24 (0.06) 0.69 0.21
NOE vs OF distance travelled cm -255 (156) -49 (145) -179 (99) -26 (95) 0.83 0.7

Rotarod

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Variables shown are: RR naive fall time, Fall time on accelerating rotarod (log₁₀ s), naive performance in session 1. RR learning, Learning on rotarod, measured as increase in fall time per trial (s/trial) in session 1. RR memory, Memory on rotarod, measured as excess fall time at middle of session 2 relative to middle of session 1.

 

Variable Units Wildtype M (n=13) Wildtype F (n=13) Mutant M (n=14) Mutant F (n=21) P(sex×mutation) P(mutation)
RR naive fall time log10 s 0.96 (0.11) 0.9 (0.08) 0.77 (0.09) 1.07 (0.07) 0.04 * 0.95
RR learning s/trial 0.3 (0.7) 1.3 (0.5) 1.2 (0.7) 1.8 (0.5) 0.68 0.25
RR memory s 11.1 (7.1) 8.3 (3.4) 4.8 (2.2) 5.4 (3.6) 0.7 0.31

Fear Conditioning

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Variables shown are: Fear learning, trial effect, Fear learning, measured as extra % time freezing before third trial compared to % time freezing before first trial. Fear learning, tone effect, Fear learning, measured as increase in % time freezing due to third tone compared to increase in % time freezing due to first tone. Contextual memory, mean, Contextual memory, measured as difference in % time freezing during first 120 s re-exposure to the box compared to first 120 s in the box on previous day. Contextual memory, change, Contextual memory, measured as increase in % time spent freezing from first time bin of 30 s to fourth bin of 30 s during 120 s re-exposure to the box. Cued memory, mean, Cued memory, measured as increase in % time spent freezing during 120 s of tone re-exposure compared to increase in % time spent freezing during initial tone on previous day. Cued memory, change, Cued memory, measured as increase in % time spent freezing from first time bin of 30 s to fourth bin of 30 s during 120 s re-exposure to the tone.

 

Variable Units Wildtype M (n=13) Wildtype F (n=13) Mutant M (n=14) Mutant F (n=21) P(sex×mutation) P(mutation)
Fear learning, trial effect % freezing 53.6 (7.2) 57.5 (7.3) 51 (4.2) 52.1 (6.2) 0.83 0.53
Fear learning, tone effect % freezing -0.9 (6) -4.8 (5.2) 4.9 (5.2) 1.5 (4.3) 0.97 0.25
Contextual memory, mean % freezing 41.9 (5.6) 42.8 (5.6) 43.3 (5.6) 46.1 (4.3) 0.86 0.65
Contextual memory, change % freezing 30.4 (9.6) 29.4 (7.6) 36.7 (6.5) 23.9 (5.6) 0.42 0.99
Cued memory, mean % freezing -4.6 (7.5) 4.2 (7.2) 0.8 (3) 12.5 (3.9) 0.79 0.21
Cued memory, change % freezing -1.2 (8.7) 0.4 (6.3) -6.6 (4.8) -3.7 (3.3) 0.91 0.41

Overview

Mutant slices showed little overall electrophysiological difference from wildtype slices. With heterozygous genotype and only one electrophysiological variable significantly affected in mutant slices, this mutation was deemed haplosufficient. Note that in the theta burst analysis, the tenth burst is analysed as representative of individual burst amplitude.

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The G2CMine data warehouse provides slice group summaries and individual mouse observations from the SAP97 knock-out line phenotyping.

 

Variables shown are: Max fEPSP amplitude, Maximum field excitatory postsynaptic potential (fEPSP) amplitude. PPF at 50ms, ampl ratio, Paired pulse facilitation (PPF), pulses separated by 50ms, amplitude ratio. Burst 1, PPF at 10ms, ampl ratio, Paired pulse facilitation (PPF), pulses separated by 10ms, amplitude ratio, observed during first two pulses of the first 100Hz burst during theta-burst stimulation. Burst 1, EPSP3 depr, ampl ratio, Depression observed in third fEPSP relative to the second fEPSP of the first 100Hz burst, amplitude ratio. Burst 1, ave amplitude, Average amplitude of four fEPSPs in first burst. Burst 2, ave amplitude, Average amplitude of four fEPSPs in second burst. Burst 3, ave amplitude, Average amplitude of four fEPSPs in third burst. Burst 4, ave amplitude, Average amplitude of four fEPSPs in fourth burst. Burst 5, ave amplitude, Average amplitude of four fEPSPs in fifth burst. Burst 6, ave amplitude, Average amplitude of four fEPSPs in sixth burst. Burst 7, ave amplitude, Average amplitude of four fEPSPs in seventh burst. Burst 8, ave amplitude, Average amplitude of four fEPSPs in eighth burst. Burst 9, ave amplitude, Average amplitude of four fEPSPs in ninth burst. Burst 10, ave amplitude, Average amplitude of four fEPSPs in tenth burst. Burst 2-4 ave amplitude vs Burst 1, Facilitation observed in average amplitude of bursts 2-4, relative to average amplitude of burst 1. Burst 8-10 ave amplitude vs Burst 2-4, Depression observed in average amplitude of bursts 8-10, relative to average amplitude of bursts 2-4. LTP vs PTP, amplitude ratio, Reduction in potentiation from immediately after theta-burst stimulation to one hour later, fEPSP amplitude ratio. LTP based on amplitude, Long term potentiation, ratio of amplitudes of fEPSPs in test pathway and control pathway.

 

Variable Units Wildtype slices (animals) Wildtype mean (SEM) Mutant slices (animals) Mutant mean (SEM) P(animals) P(mutation)
Max fEPSP amplitude µV 17 (5) 2890 (170) 14 (6) 2860 (130) 0.37 0.89
PPF at 50ms, ampl ratio % 17 (5) 180.2 (5.9) 14 (6) 176.2 (8.5) 0.45 0.71
Burst 1, PPF at 10ms, ampl ratio % 17 (5) 200 (12) 14 (6) 198 (13) 0.58 0.93
Burst 1, EPSP3 depr, ampl ratio % 17 (5) 69.9 (3.9) 14 (6) 70.8 (4.3) 0.37 0.89
Burst 1, ave amplitude µV 17 (5) 1269 (59) 14 (6) 1222 (52) 0.83 0.47
Burst 2, ave amplitude µV 17 (5) 1888 (126) 14 (6) 1776 (93) 0.55 0.49
Burst 3, ave amplitude µV 17 (5) 2190 (150) 14 (6) 2070 (120) 0.64 0.5
Burst 4, ave amplitude µV 17 (5) 2340 (160) 14 (6) 2240 (130) 0.67 0.6
Burst 5, ave amplitude µV 17 (5) 2320 (160) 14 (6) 2260 (130) 0.7 0.74
Burst 6, ave amplitude µV 17 (5) 2290 (150) 14 (6) 2240 (130) 0.72 0.79
Burst 7, ave amplitude µV 17 (5) 2230 (150) 14 (6) 2180 (130) 0.72 0.78
Burst 8, ave amplitude µV 17 (5) 2160 (150) 14 (6) 2130 (130) 0.7 0.89
Burst 9, ave amplitude µV 17 (5) 2090 (150) 14 (6) 2080 (130) 0.74 0.96
Burst 10, ave amplitude µV 17 (5) 2050 (150) 14 (6) 2040 (120) 0.75 0.93
Burst 2-4 ave amplitude vs Burst 1 % 17 (5) 168.1 (4.9) 14 (6) 168.8 (8.7) 0.31 0.95
Burst 8-10 ave amplitude vs Burst 2-4 % 17 (5) 97.5 (1.1) 14 (6) 101.7 (1.8) 0.93 0.016 *
LTP vs PTP, amplitude ratio % 17 (5) 79.4 (1.4) 14 (6) 74.5 (1.5) 0.051 0.11
LTP based on amplitude % 17 (5) 175.1 (6.9) 14 (6) 176.8 (10) 0.71 0.87

Basal Synaptic Transmission

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Variables shown are: Max fEPSP amplitude, Maximum field excitatory postsynaptic potential (fEPSP) amplitude.

 

Variable Units Wildtype slices (animals) Wildtype mean (SEM) Mutant slices (animals) Mutant mean (SEM) P(animals) P(mutation)
Max fEPSP amplitude µV 17 (5) 2890 (170) 14 (6) 2860 (130) 0.37 0.89

Paired Pulse Facilitation

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Variables shown are: PPF at 50ms, ampl ratio, Paired pulse facilitation (PPF), pulses separated by 50ms, amplitude ratio.

 

Variable Units Wildtype slices (animals) Wildtype mean (SEM) Mutant slices (animals) Mutant mean (SEM) P(animals) P(mutation)
PPF at 50ms, ampl ratio % 17 (5) 180.2 (5.9) 14 (6) 176.2 (8.5) 0.45 0.71

Theta Burst Stimulation

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Variables shown are: Burst 1, PPF at 10ms, ampl ratio, Paired pulse facilitation (PPF), pulses separated by 10ms, amplitude ratio, observed during first two pulses of the first 100Hz burst during theta-burst stimulation. Burst 1, EPSP3 depr, ampl ratio, Depression observed in third fEPSP relative to the second fEPSP of the first 100Hz burst, amplitude ratio. Burst 1, ave amplitude, Average amplitude of four fEPSPs in first burst. Burst 2, ave amplitude, Average amplitude of four fEPSPs in second burst. Burst 3, ave amplitude, Average amplitude of four fEPSPs in third burst. Burst 4, ave amplitude, Average amplitude of four fEPSPs in fourth burst. Burst 5, ave amplitude, Average amplitude of four fEPSPs in fifth burst. Burst 6, ave amplitude, Average amplitude of four fEPSPs in sixth burst. Burst 7, ave amplitude, Average amplitude of four fEPSPs in seventh burst. Burst 8, ave amplitude, Average amplitude of four fEPSPs in eighth burst. Burst 9, ave amplitude, Average amplitude of four fEPSPs in ninth burst. Burst 10, ave amplitude, Average amplitude of four fEPSPs in tenth burst. Burst 2-4 ave amplitude vs Burst 1, Facilitation observed in average amplitude of bursts 2-4, relative to average amplitude of burst 1. Burst 8-10 ave amplitude vs Burst 2-4, Depression observed in average amplitude of bursts 8-10, relative to average amplitude of bursts 2-4.

 

Variable Units Wildtype slices (animals) Wildtype mean (SEM) Mutant slices (animals) Mutant mean (SEM) P(animals) P(mutation)
Burst 1, PPF at 10ms, ampl ratio % 17 (5) 200 (12) 14 (6) 198 (13) 0.58 0.93
Burst 1, EPSP3 depr, ampl ratio % 17 (5) 69.9 (3.9) 14 (6) 70.8 (4.3) 0.37 0.89
Burst 1, ave amplitude µV 17 (5) 1269 (59) 14 (6) 1222 (52) 0.83 0.47
Burst 2, ave amplitude µV 17 (5) 1888 (126) 14 (6) 1776 (93) 0.55 0.49
Burst 3, ave amplitude µV 17 (5) 2190 (150) 14 (6) 2070 (120) 0.64 0.5
Burst 4, ave amplitude µV 17 (5) 2340 (160) 14 (6) 2240 (130) 0.67 0.6
Burst 5, ave amplitude µV 17 (5) 2320 (160) 14 (6) 2260 (130) 0.7 0.74
Burst 6, ave amplitude µV 17 (5) 2290 (150) 14 (6) 2240 (130) 0.72 0.79
Burst 7, ave amplitude µV 17 (5) 2230 (150) 14 (6) 2180 (130) 0.72 0.78
Burst 8, ave amplitude µV 17 (5) 2160 (150) 14 (6) 2130 (130) 0.7 0.89
Burst 9, ave amplitude µV 17 (5) 2090 (150) 14 (6) 2080 (130) 0.74 0.96
Burst 10, ave amplitude µV 17 (5) 2050 (150) 14 (6) 2040 (120) 0.75 0.93
Burst 2-4 ave amplitude vs Burst 1 % 17 (5) 168.1 (4.9) 14 (6) 168.8 (8.7) 0.31 0.95
Burst 8-10 ave amplitude vs Burst 2-4 % 17 (5) 97.5 (1.1) 14 (6) 101.7 (1.8) 0.93 0.016 *

Long Term Potentiation

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Variables shown are: LTP vs PTP, amplitude ratio, Reduction in potentiation from immediately after theta-burst stimulation to one hour later, fEPSP amplitude ratio. LTP based on amplitude, Long term potentiation, ratio of amplitudes of fEPSPs in test pathway and control pathway.

 

Variable Units Wildtype slices (animals) Wildtype mean (SEM) Mutant slices (animals) Mutant mean (SEM) P(animals) P(mutation)
LTP vs PTP, amplitude ratio % 17 (5) 79.4 (1.4) 14 (6) 74.5 (1.5) 0.051 0.11
LTP based on amplitude % 17 (5) 175.1 (6.9) 14 (6) 176.8 (10) 0.71 0.87
© G2C 2014. The Genes to Cognition Programme received funding from The Wellcome Trust and the EU FP7 Framework Programmes:
EUROSPIN (FP7-HEALTH-241498), SynSys (FP7-HEALTH-242167) and GENCODYS (FP7-HEALTH-241995).

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