KO mice

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KO mice

• Gyorgy Bagdy
• 26 04 2008

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• KO mice gene(s) deleted
• null mutant homozygous -/-
• heterozygote /-
• wild type /
• Transgenic mice extra gene(s) added
• a normal gene is inserted into the mouse genome
  overexpression
• a new gene is added that is not normally present
  in the mouse genome modell of diseases

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• Knock out of a gene
• altered function
• the encoded protein of the targeted gene carries
  the function?
• Phenotype biochemical, anatomical, physiological
  and behavioral characteristics.

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Standardized and special tests

• Special
• tests adequately to the mutations
• flexible and creative analysis of behaviour

• Standardized
• compare the results from different laboratories
• tests are rigid and unalterable

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Test batteries

• The goal of preliminary observations is to
  eliminate artifacts.
• e.g. blind mouse is useless for visual
  discrimination tasks
• 
  Which battery?
• The behaviour of the mouse is simple to describe
  them with several tests?
• The behaviour of the mouse is complex, the brain
  of the mouse is complex to show various
  behavioural traits?

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Example 1.

• Hypothesis the CamKII (a gene coding one of the
  parts of the calmodulin-dependent kinase) protein
  plays a role in learning
• Test Morris Water Maze test
• CamKII null-mutant mice fail to learn
• Turned out later these mice are abnormally shy,
  over anxious, they were unable to learn because
  they were frightened
  
  
  Silva et
  al. 1992

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Example 2.

• Developing anxiolytic drugs
• Hypothesis mGluR8 null-mutant mice are anxious,
  its increased in a box being higly illuminated
• Anxiolitic drugs were tested
• Turned out later the ICR mouse strain (used to
  develop the mutants) carries a gene causing
  retina degeneration, so mice were almost blind
• 
  
  Linden és mtsi. 2002
• 
  
  
  

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1. General appearance

• healthy mouse well-groomed
• sick mouse often stops grooming yellow, grey,
  sticky fur indicates insufficient grooming, bald
  patches reflect aberrant grooming
• sparse, short, or absent whiskers inadequate
  diet, aberrant self-grooming, excessive whisker
  barbering by a cagemate
• pink ear pinna and footpads a sign of good
  health hypothermia produces pale or purple
• very large or small body size abnormal growth,
  feeding or metabolism

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2. Body weight

• mice are weighed on animal balances
• if the gene under investigation is relavant to
  feding behaviors, then it may be useful to
  measure body weight daily
• 3. Body temperature
• measured in mice by a rectal thermistor
• 4. Posture and gait
• normal mice show a hunched posture, with feet
  tucked under the body when resting
• they locomote with all four feet moving and ears
  and whiskers twitching
• during pauses the head and whiskers may move
  quickly

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5. Home cage activity patterns

• videotaping several 24-hour periods
• mice are nocturnal spieces, home cage activity is
  higher during the dark phase
• exploratory activity occurs spontaneously, bouts
  of exploration alternate with boust of resting
  and sleeping
• mating occures primarily during the dark phase
• sleeping patterns generally resemble a group
  huddle, on top of each other
• 6. Abnormal spontaneous behaviors
• the mouse is placed in an empty cage for
  observation
• e.g. wild running, excessive grooming and
  stereotyped sniffing, frozen immobility
• 3 min observation period

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7. Neurological reflexes

• righting reflex evaluated by turning the mouse
  onto its back
• postural reflex evaluated by placing the mouse
  in an empty cage and shaking the cage the
  extention of all four legs to maintain a balanced
  position
• eye blink reflex tested by approaching the eye
  with the tip of a clean cotton swab
• ear twitch reflex tested by touching the ear
  with the tip of a clean cotton swab
• whisker-orienting reflex tested by lightly
  brushing the whiskers of a freely moving animal
  with a small paint brush

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8. Vision

• pupil constriction and dilation flashlight is
  directed at the eye
• 9. Olfactory
• detect anosmia a strong, pleasant odor is
  painted onto the side of a clean, bare test cage
• 10. Tactile
• measured with Von Frey hairs
• reflex responses to pain are quantitated with
  tail flick and hot plate

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Moser Neurobehavioral Toxicology Test Battery

• Excitability
• Ease of removal
• Handling reactivity
• Arousal
• Clonic movements
• Tonic movements
• Physiological measures
• Body weight
• Body temperature
• Piloerection

• Activity
• Rearing
• Motor activity counts
• Home cage posture
• Sensorimotor
• Tail-pinch response
• Click response
• Touch response
• Approach response

• Autonomic
• Lacrimation
• Salivation
• Palpebral closure
• Pupil response
• Urination
• Neuromuscular
• Gait score
• Righting reflex
• Forelimb grip strenght
• Hindlinb grip strenght
• Landing foot posture

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SHIRPA (Rogers et al. 1997)

• Primary Screen
• Behavioral observation profile

Secondary Screen Locomotor
activity Food and water intake Balance and
coordination Analgesia Histology Biochemistry
Tertiary Screen
(complex behavior)
Anxiety Learning and
memory EMG, EEG, MR
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Sleep-wake and circadian rhythm in serotonin
transporter KO and control mice
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Effect of the deletion of the 5-HTT, 5-HT1A and
5-HT1B receptor (a) genes and the (b)
pharmacological blockade (grey graphs) on REM
sleep
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Effect of the deletion of 5-HT2A receptor gene
and the receptors pharmacological blockade on
N-REM and REM sleep
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The amount of 24h REM sleep in adult 5-HTT KO
mice, and its normalization after neonatal 5-HT1A
receptor antagonist or 5-HT neurotoxin treatment
(5-19 and 4-32 days)
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Summary of the effects on sleep of gene deletion
vs. pharmacological inhibition of various
components of serotonergic neurotransmission
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