Genetics of lab animals

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Genetics of lab animals

• Dag Eide
• Oslo 16.10.2003

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Motivation

• Reducing experimental errors
• Outbred versus inbreds
• Genetic-environment interactions
• in standard experiments
• importance of design
• Genetic studies
• GM animals, mutants, special strains
• Breeding techniques

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Standardization

• Standardization of the animal
• Standardization of environment
• Reproducibility

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Reproducibility

• Reducing experimental errors due to uncontrolled
  genetic variation
• Refinement

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Genetic qualities of small rodents

• Outbred stocks
• Inbred strains
• variations

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Outbred stocks

• closed colony
• minimal selection
• lt 1 increase in the inbreeding coefficient pr.
  generation
• ?F 1/8nf 1/8Nm

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Outbred stocks

• When to use
• Production. (antibodies, cell cultures, etc)
• When inbreds could lead to poorer results.
• Selection experiments
• Experiments where frequencies of undefined
  characteristic in an undefined population is
  interesting
• Standardized safety testing
• Cheap animals (???)
• Inbreds are not available (most species)
• Examples nomenclature
• MolWIST
• BomNMRI

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Outbred stocks - problems

• Genetic drift gene frequencies will change in
  closed colonies
• Use same source of animals
• Some breeders cooperate
• Wistar ? Swiss
• BUT Wistar ? Wistar is also true
• Should be replaced by inbreds if possible
• (mosaic population)

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Isogenic animals

• monozygotic twins
• Clones - gynogenetic animals
• Inbreds ( 98)
• Inbred F1 hybrids

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Inbred strains

• When
• quantitive traits are measured
• Genetic background may interfere with results
• Almost always!

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Inbred strains - definitions

• gt 20 generations brother-sister matings

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Innavl i praksis
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Inbred strains - nomenclature

• gt 1000 inbred mouse strains
• BALB/cJ
• CBA/Bom
• WKY/Mol

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Inbreds - phenotype differences

• Pentobarbital (75 mg/kg) sleeping time
• minutes
• DBA/2J 475
• C57BL/6J 235
• ICR 101
• Claasen (1994)

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Species inbred

• Hamster
• gerbil
• guinea pig
• Chicken
• Amphibians
• fish (gynogenesis also)

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Inbreds - problems

• Inbreeding depression (F2-F8)
• Mutations
• new variants
• Rest heterozygozity

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Rest heterozygozity
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Maintaining your own inbreds

• Buy founders
• Mate randomly 5-10 generations
• Or perform genetic QC
• Select best breeders
• Replenish population with original founders

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F1-hybrids

• Crossing 2 inbred strains
• isogenic
• Heterosis effect robust, viable, less sensitive,
  
• expensive
• Should be used more!

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Comparison
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Coisogenic strains

• Strain that differs from parental strain in ony
  one gene / locus
• Historically mutants
• Ex. Balb/c-nu
• GM animals
• Created by backcrossing
• Often maintained by backcrossing

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Challenges for animal units

• maintain protocols
• - skilled personell
• - avoid mix up
• Backcrossing nomenclature - N-tag
• E.g. Tg(Vr-1b)JN3
• Keep tight timing schedules
• Breeding takes time
• Researchers dont have too much of it..

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Genetic Quality control
Test methods

• DNA fingerprinting by PCR
• Jackson laboratories 108 PCR markers for
  standard strains
• SSLP - simple sequence length polymorphisms
• SNPs - single nucleotide polymorphisms - are not
  readily available

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Genotyping protocols

• 
  

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Cryopreservation

• freezing thawing of embryo
• preserve rare strains
• Jax will still supply you
• preserve inbreds to be able to check for genetic
  drift
• Transfer of strains

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Inbred animals are preferred

• Genetically defined animals will increase
  reproducibility
• In ANY experiment
• Use one source (still)
• Describe your animals correctly in MM

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How GM rodents started

• Nature 1982 300(5893)611-5

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Science 14 april 2000

• Increase in animal use
• Good animal facilities demanded by the best
  researchers

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What is a GM animal?

• animals modified by adding, removing or replacing
  original DNA using molecular biology
• Mutants are not included, even if they have the
  same application

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Why mice?

• Many years ahead
• Plenty of inbred strains
• Many genetic markers known
• short generation interval
• Homologous genes described i inbreds and mutants
• ES celler established for several inbred lines
• Injection in pronucleus not too hard
• Easy surgery

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Necessary reading
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Transgenic animals

• DNA sequence introduced in the mouse genome
• Microinjection in pronucleus
• retrovirus
• embryonic stem cells (ES cells)

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Injeksjon i pronucleus
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ES - what it is

• from mouse embryos
• Maintain ability to form embryos after several
  passages in culture
• May be selected for presence of introduced DNA in
  vitro (e.g. neomycin resistance gene)
• Can be introduced into already existing embryo
• Leads to mosaic animals - part TG/KO/KIN, part
  wild type

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ES cells - general

• strain 129 most common
• C57BL/6 recipient (most common)
• technique
• knock-in, knock-out, point mutations
• a lot of work, integration may be random

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KO
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Injeksjon av ES
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GM animals mutants

• Genetic background IMPORTANT
• MIN-mutation on B6/J gt spontaneous tumors
• MIN on AKR/J gt no tumors
• Increased consciousness the last years about this
• Mutants
• spontaneous
• induced
• Jackson Labs
• Mammalian genetics unit (MRC, UK)

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Problems w/GM-animals

• GM mice often hard to keep
• Disease - genetic defcts
• (sub)lethal defects
• Infections all too common
• Source of animals?
• Jackson labs cleans all strains before entry
• FHI isolator og Microbiologic control

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Maintenance and breeding in a ordinary animal unit

• backcrossing
• maintenance
• Producing double-GM animals
• Crossing of strains
• Record keeping (again)
• Silvers book is available at www.informatics.jax.
  org/silver

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How to get GM-animals?

• Commercial breeders
• Hi-quality
• Research groups
• Universities, institutes
• Jackson labs
• patented?
• May you breed the animals yourself?

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GM / mutant maintenance

• Requires a High standard animal unit
• Quarantene facilities
• often not SPF
• protect animals and caretakers/researchers
• expensive models
• often unique animals
• Labor intensive
• ca 10 X SPF aanimals
• Even more sensitive to environmental variations

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Legal requirements

• Helsedirektoratet must approve the facilities
• --- and should be notified for every new GMO in
  use in the facility
• http//bioteknologi.helsetilsynet.no/gmo
• Acb_at_shdir.no
• Direktoratet for naturforvaltning
• Notification about import
• Notification about transport
• www.dirnat.no

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Xenotransplants

• Check
• http//Odin.dep.no
• NOU Xenotransplantasjon

Full URL http//odin.dep.no/hd/norsk/publ/utredni
nger/NOU/014001-020007/index-dok000-b-n-a.html