Comprehensive Behavioral and Molecular Characterization of a New Knock-In Mouse Model of Huntington's Disease: zQ175 methods
Aim. Evidence-backed execution summary for Comprehensive Behavioral and Molecular Characterization of a New Knock-In Mouse Model of Huntington's Disease: zQ175 methods from Comprehensive Behavioral and Molecular Characterization of a New Knock-In Mouse Model of Huntington's Disease: zQ175.
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This experiment, in seven questions
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Shopping and prep list
What do I need before I start?
mouse
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Total RNA extraction
reagent used in the protocol.
- Use
- Tissues were homogenized 2 × 1 min at 25 Hz in 750 µL of QIAzol Lysis Reagent (Cat # 79306, Qiagen, Valencia, CA) with TissueLyser (Qiagen, Valencia, CA) and 5 mm stainless steel beads (Cat # 69989, Qiagen, Valencia, CA). Once tissues were disrupted, samples were allowed to incubate at room temperate for 5 m...
Motor Abnormalities
Motor abnormalities, cardinal features of the human condition, are also apparent in many HD mouse models,,,,,. We used four behavioral methods to evaluate motor decline; rotarod performance, open field activity, climbing behavior, and the PhenoCube® system. Testing in the diurnal dark phase when spontaneo...
- Use
- Motor abnormalities, cardinal features of the human condition, are also apparent in many HD mouse models,,,,,. We used four behavioral methods to evaluate motor decline; rotarod performance, open field activity, climbing behavior, and the PhenoCube® system. Testing in the diurnal dark phase when spontaneo...
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First confirmation
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This page is backed by a publishable Replication Data Ledger package with zero critical source-verification issues.
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Open the source paper before finalizing run-specific details.
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Open quote workflowStep-by-step procedure
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Materials and Methods
Homozygous, heterozygous and wild type (WT) mice were generated by crossing heterozygous zQ175 mice on a C57B/l6J background. Animals were ear notched at around 10-15 days. Mice were weaned and implanted with RFID electronic chips (DataMars, OH) for identification at around 21 days. The average CAG repeat length in the primary test cohort was 186.2±15 (SEM = 1.4) for the heterozygous and 188.7±12 (SEM = 1.0) for the homozygous animals. Genotyping and CAG repeat count were determined by Laragen Inc. (Culver City, CA), from PCR of tail snips taken at 10-15 days of age. CAG repeat length and range in the other cohorts examined were comparable.
Materials and Methods
Tissue was collected at three timepoints, at 12, 18 and 41 weeks of age, with an n = 5-12 per group. Tissue from the 12 and 18 week timepoints was collected from behaviorally naïve satellite groups, while the 41 week collection was taken from animals evaluated in the PhenoCube at 37 weeks (see cohort 3 in ).
Total RNA extraction
Tissues were homogenized 2 × 1 min at 25 Hz in 750 µL of QIAzol Lysis Reagent (Cat # 79306, Qiagen, Valencia, CA) with TissueLyser (Qiagen, Valencia, CA) and 5 mm stainless steel beads (Cat # 69989, Qiagen, Valencia, CA). Once tissues were disrupted, samples were allowed to incubate at room temperate for 5 minutes. For the RNA extraction procedure, the manufacturer's protocol for RNeasy 96 Universal Tissue Kit (Cat # 74881, Qiagen, Valencia, CA) for RNA isolation was followed. Briefly, 150 µL of Chloroform (Cat # C2432, Sigma-Aldrich, St. Louis, MO) was added and samples were shaken vigorously for 15 seconds followed by 3 minutes incubation at room temperature. The aqueous phase was separated from the organic phase by centrifugation at 6,000 × g (Beckman Coulter Avanti J-30I), at 4°C for 15 minutes. The aqueous phase was then transferred to a new 96-well blo...
Motor Abnormalities
Motor abnormalities, cardinal features of the human condition, are also apparent in many HD mouse models,,,,,. We used four behavioral methods to evaluate motor decline; rotarod performance, open field activity, climbing behavior, and the PhenoCube® system. Testing in the diurnal dark phase when spontaneous activity was higher (illustrated by the PhenoCube data) showed genotype-dependent deficits that otherwise might not have been observed. Whilst the basic locomotor and rearing abnormalities detected in this new line have an early onset and are robust, especially in the homozygotes, the deficits in those parameters did not seem to progress with age (at least as examined, up to 36 weeks). It is possible that these mice might progress at older ages, though age-related decline in performance of the WT mice may complicate analysis.
Measurement outputs
What raw and processed outputs should exist?
Homozygous, heterozygous and wild type (WT) mice were generated by crossing heterozygous zQ175 mice on a C57B/l6J background. Animals were ear notched at around 10-15 days...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
An alpha level of.05 was selected for all inferential statistics. Repeated measures analysis (age as dependent factor) was carried out with SAS (SAS Institute Inc.) using Mixed...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Motor abnormalities, cardinal features of the human condition, are also apparent in many HD mouse models,,,,,. We used four behavioral methods to evaluate motor decline; r...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Analysis plan
How should the outputs become interpretable results?
Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
inferred from protocolPreprocessing / cleaning
Homozygous, heterozygous and wild type (WT) mice were generated by crossing heterozygous zQ175 mice on a C57B/l6J background.
from paperScoring or quantification
Quantify the primary readouts for this experiment: Homozygous, heterozygous and wild type (WT) mice were generated by crossing heterozygous zQ175 mice on a C57B/l6J background. Animals were ear notched at around 10-15 days...; An alpha level of.05 was selected for all inferential statistics. Repeated measures analysis (age as dependent factor) was carried out with SAS (SAS Institute Inc.) using Mixed...; Motor abnormalities, cardinal features of the human condition, are also apparent in many HD mouse models,,,,,. We used four behavioral methods to evaluate motor decline; r....
from paperStatistical comparison
Homozygous, heterozygous and wild type (WT) mice were generated by crossing heterozygous zQ175 mice on a C57B/l6J background. Animals were ear notched at around 10-15 days...; An alpha level of.05 was selected for all inferential statistics. Repeated measures analysis (age as dependent factor) was carried out with SAS (SAS Institute Inc.) using Mixed...
from paperReporting output
Report representative outputs alongside summary comparisons for Homozygous, heterozygous and wild type (WT) mice were generated by crossing heterozygous zQ175 mice on a C57B/l6J background. Animals were ear notched at around 10-15 days..., An alpha level of.05 was selected for all inferential statistics. Repeated measures analysis (age as dependent factor) was carried out with SAS (SAS Institute Inc.) using Mixed..., Motor abnormalities, cardinal features of the human condition, are also apparent in many HD mouse models,,,,,. We used four behavioral methods to evaluate motor decline; r....
inferred from protocolStructured statistical methods
Homozygous, heterozygous and wild type (WT) mice were generated by crossing heterozygous zQ175 mice on a C57B/l6J background. Animals were ear notched at around 10-15 days...; An alpha level of.05 was selected for all inferential statistics. Repeated measures analysis (age as dependent factor) was carried out with SAS (SAS Institute Inc.) using Mixed...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (4)
Homozygous, heterozygous and wild type (WT) mice were generated by crossing heterozygous zQ175 mice on a C57B/l6J background. Animals were ear notched at around 10-15 days. Mice were weaned and implanted with RFID electronic chips (DataMars, OH) for identification at around 21 days. The average CAG repeat length in the primary test cohort was 186.2±15 (SEM = 1.4) for the heterozygous and 188.7±12 (SEM = 1.0) for the homozygous animals. Genotyping and CAG repeat count were determined by Laragen Inc. (Culver City, CA), from PCR of tail snips taken at 10-15 days of age. CAG repeat length and range in the other cohorts examined were comparable.
Tissue was collected at three timepoints, at 12, 18 and 41 weeks of age, with an n = 5-12 per group. Tissue from the 12 and 18 week timepoints was collected from behaviorally naïve satellite groups, while the 41 week collection was taken from animals evaluated in the PhenoCube at 37 weeks (see cohort 3 in ).
Tissues were homogenized 2 × 1 min at 25 Hz in 750 µL of QIAzol Lysis Reagent (Cat # 79306, Qiagen, Valencia, CA) with TissueLyser (Qiagen, Valencia, CA) and 5 mm stainless steel beads (Cat # 69989, Qiagen, Valencia, CA). Once tissues were disrupted, samples were allowed to incubate at room temperate for 5 minutes. For the RNA extraction procedure, the manufacturer's protocol for RNeasy 96 Universal Tissue Kit (Cat # 74881, Qiagen, Valencia, CA) for RNA isolation was followed. Briefly, 150 µL of Chloroform (Cat # C2432, Sigma-Aldrich, St. Louis, MO) was added and samples were shaken vigorously for 15 seconds followed by 3 minutes incubation at room temperature. The aqueous phase was separated from the organic phase by centrifugation at 6,000 × g (Beckman Coulter Avanti J-30I), at 4°C for 15 minutes. The aqueous phase was then transferred to a new 96-well block and total RNA was precipitated with an equal volume of 70% ethanol. Total volume was then transferred to an RNeasy 96-well plate, followed by centrifuging at 6,000 × g (Beckman Coulter Avanti J-30I), at room temperature for 4 minutes. Total RNA bound to column membranes was then treated with R...
Motor abnormalities, cardinal features of the human condition, are also apparent in many HD mouse models,,,,,. We used four behavioral methods to evaluate motor decline; rotarod performance, open field activity, climbing behavior, and the PhenoCube® system. Testing in the diurnal dark phase when spontaneous activity was higher (illustrated by the PhenoCube data) showed genotype-dependent deficits that otherwise might not have been observed. Whilst the basic locomotor and rearing abnormalities detected in this new line have an early onset and are robust, especially in the homozygotes, the deficits in those parameters did not seem to progress with age (at least as examined, up to 36 weeks). It is possible that these mice might progress at older ages, though age-related decline in performance of the WT mice may complicate analysis.
Machine-readable layer
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