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Wednesday, July 14, 2010

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The Mouse as Animal Model

1. Genetic manipulation is possible Transgenic mice Gene targeted mice

2. Three month generation time3. Behavioural test are available4. Physiology is well established


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candidate gene phenotypic analysis

Reverse Genetic Approach


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1) Pharmacological study

Drug treated versus naive mice naive

Comparative Phenotyping


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1) Pharmacological study

Drug treated versus naive mice naive

2) Genetically modified mouse line

Genetic manipulated versus WT mice

Comparative Phenotyping


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First question for setting up theexperimental design

Genetic Background

(Mouse strain)

C57Bl6


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First question for setting up theexperimental design

Genetic Background

(Mouse strain)

C57Bl6

C57Bl6/J C57Bl6/N


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C57BL/6J, C57BL/6N, and C57BL/6C mice in a behavior test battery.

700–2,200 mice in total.

Significant differences among the substrains were found in the results of various behavioral tests, including the open field, rotarod, elevated plus maze, prepulse inhibition, Porsolt forced swim, and spatial working memory version of the eight-arm radial maze.

Small genetic differences may have a great influence on behavioral phenotypes. Thus, the genetic background of different substrains should be carefully chosen, equated, and considered in the interpretation of mutant behavioral phenotypes


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Gene targeted mice

ES cell line SV129

X

C57Bl6/J


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Gene targeted mice

ES cell line SV129

X

C57Bl6/J

F1 hybridSV129- C57Bl6/J


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Gene targeted mice

ES cell line SV129

X

C57Bl6/J

C57Bl6/J

20backcrosses(3 years)F1 hybrid

SV129- C57Bl6/J


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F1 hybrid C57Bl6/J20

backcrosses(3 years)SV129- C57Bl6/J

Solid phenotypes Phenotypes on C57Bl6/X background


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Transgenic mice

multiple foundershave to be analysed


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Transgenic mice

multiple foundershave to be analysed

Gene-targeted mice

one founder issufficient for theanalysis


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Transgenic lines

Multiple founders have to be analyzed

Offsprings from two independent founders, with two different integration sites have to be analysed in behaviour!

Four groups of mice (10 -12 mice per group)


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Conditional transgenic miceGroup 1: Gene “ON”Group 2: Gene “Off”

Site-effect of the drug needs attention.

Transgenic lines


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Possible confounding influences of the laboratory environment were studied in several inbred strains and one null mutant

by simultaneous testing in three laboratories on a battery of sixbehaviors.

Apparatus, test protocols, and many environmental variables wererigorously equated.

Despite standardization, there were systematic differences in behavior across labs


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Reliability of behavioral phenotyping is not challenged seriously by experimenterand laboratory environment as long as appropriate standardizations are met and suitable controls are involved.

Lewejohann et al., Genes, Brain and Behavior (2006) 5: 64–72.


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Starting The Experiment

Set up multiple breedings

21 days + two month

het x wthet

wt

Transgenic


Sprengel 07/10

Starting The Experiment

Set up multiple breedings

21 days + two month

het x wthet

wt

Transgenic

het x het

KO

het

wt

Gene targeted


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The Experiment


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The Experiment

Move the mice to the facility where the behavioural experiments should be performed

single housing with toys (enriched environment)without toys

start handling each mouse 10 to 20 min per day

diet down to 85 % body weight

PRE - HANDLING


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Mouse handling prior to behavioural testing

Mice were taken out of their cage by the tail and placed on the experimenter's arm wearing a lab coat.

This was done until the mice were used to the experimenter and to the fact that they were taken out of the cage.

Wearing always the same lab coat is helpful for the habituation, because the mouse smell on the lab coat decreases the anxiety.

Before starting to train mice in appetitive motivated tasks (food reward) mice were habituated to condensed milk (diluted in water; 50/50 w/w) first in their home cage, then in an open field. Habituation to the condensed milk in the open field aimed to motivate the mouse to explore the environment for food reward.


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Data from each mouse:

AppearanceNesting scoreTail suspension: neurodegenarationSwimming test Neophobia: latency to drinkOlfactory: latency to find buried foodAccelerating Rota-Rod: latency to fall offHorizontal bar score: latency to fall offOpen field: ActivityLight-dark-box evaluation: Anxiety

General animal handling prior to behavioural testing


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Phenotyping


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newborns (P0)


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Pubs (P15-P21)


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Adults (>P42)


Adults (>P42)

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Cognitive Behaviour

Morris Water Maze

spatial reference memory


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0

10

20

30

40

50

60

70

0 1 2 3 4 5 6 7 8 9 10 11la

tency

(s)!

blocks of four trials!

GluR-A-/-!

wt!

water-maze


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T-maze Y-maze evaluation: spatial learning

Puzzel box: cognitive performance

Fear conditioning: emotional learning

Olfactory learning:

Cognitive Behaviour


Spatial Working Memory

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Spatial Reference Memory

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T-maze Y-maze


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Puzzle Box


Puzzle Box

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NeurodegenerationTail suspension:


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Emotional Learning

Passive avoidance


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Emotional Learning

Passive avoidance

Fearconditioning


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Fear conditioning


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Aquisition

Context extinction


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Olfactory Learning


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IntellicageNew Behaviour (Hans Peter Lipp)

www.newbehavior.com


http://www.newbehavior.com

http://www.newbehavior.com

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Thank you !!!!


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Building trust with your mouse is also absolutely fundamental for any training.

If the mouse feels uneasy about its surrounding and your presence, it will probably just try to escape by running along the wall for want of cover.

Such a frightened rodent won’t cross free and light area .

A stressed mouse can never concentrate

The mouse will be so nervous that it even refuses its favourite treats or other rewards outside the cage and, if ever, learn a lot more slowly.

http://mouse-agility.com

Thank you !!!!




Sprengel 07/10http://mouse-agility.comWednesday, July 14, 2010



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