Best Practices for Efficient Mouse Colony …...Best Practices for Efficient Mouse Colony Management...
Transcript of Best Practices for Efficient Mouse Colony …...Best Practices for Efficient Mouse Colony Management...
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Dominique Kagele, Ph.D.
Technical Information Services
Best Practices for Efficient Mouse Colony Management
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Overview of Today’s Presentation
2
Mouse reproduction
Factors affecting breeding performance
Data collection and good colony management
Breeding strategies
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Mouse Reproductive Milestones and Characteristics
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Gestation: 18.5 to 21 days
Litter size: 2 to 12+ pups
Weaning age: 17 to 28 days
Sexual maturity: 5 to 8 weeks
Productive breeding life: ~ 7-8 months
Silver LM. 1995. Mouse Genetics: Concepts and Applications, Oxford University Press. Available online at www.informatics.jax.org/silver
8 day old NOD/ShiLtJ pups (001976)
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Breeding Performance: Strain Background Effects
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Postnatal defects: C57BL/6J
Behavior: SJL/J
Fecundity: NOD/ShiLt, FVB/NJ
Hybrid vigor: F1, F2 hybrids
4 week old C57BL/6J mouse with hydrocephalus
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Breeding Performance: Mutation & Transgene Effects
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Embryonic lethality
Infertility or subfertility Mammary function (lactation) Abnormal behavior o Poor mothering instinct o Aggression
Shortened breeding life span o Tumor development (e.g. Trp53tm1Tyj) o Neurodegeneration (e.g. ALS, Huntington’s)
B6CBA-Tg(HDexon1)62Gpb/3J (006494)
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Breeding Performance: Environmental Effects
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Temperature
Light cycle and intensity
Noise and vibrations (construction, equipment)
Handling (overhandling; caretaker changes)
Odors (toxic fumes, perfume)
Nutrition
Health status
Season
www.jax.org/jaxmice/support/husbandry/room-conditions
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Mouse Reproduction Can Change With the Seasons
7 Breeding & Rederivation Services |
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Data Collection & Record Keeping
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Critical for successful colony management
Improve efficiency
Detect problems/mutations early
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Record Keeping: Pedigree Book
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Important information includes: Strain data o Nomenclatureo Genetic background o History Unique animal numbers
Pedigree information o Parentage (dam and sire) & date mated o Litter number, birth dates & pups born o Weaned pups
- Number, wean:born - Gender - Genotypes (if applicable)
o Generation number
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Record Keeping: Generation Number
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N = Number backcross generation(s)
F = Filial (sister x brother) generation(s)
p = cryopreserved
+ = separates generation information prior to importation
? = unknown generation number
Examples:
www.jax.org/jaxmice/support/husbandry/definitions
N6F12 + F8 ? + N10F5 N4F1 + N6F9
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JAX Colony Management System (JCMS)
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Create mating & litter records
Track pedigrees and animal status
Record genotypes
Organize experimental data
Prints cage cards
Advanced database queries & data export www.jax.org/jcms
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Record Keeping Tips
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Use pre-printed cards or labels
Use different colors
o Adjacent strains o Matings vs. weanlings
Separate strains with similar nomenclature
Keep cages from a single strain together
o Breeders and weanlings
Keep records in multiple locations
Save ALL cage cards
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Mouse Identification
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Neonates
o Tattoo (FDA approved pigment) o Toe clip
Weanlings and adults
o Ear notch o Ear tag o Micro-chip implant o JAXTag™
www.jax.org/jaxmice/support/husbandry/identification
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Ear Notching
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Example: Mouse #53 }
Numbering system from 1 to 99
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Colony Management Tips
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Mate early (6-12 weeks)
Choose breeders carefully o Avoid selection pressure
Replace breeders on a rotation (monthly) o Mixed-age colony breeds more consistently o Requires having young breeders available
Replace non-productive breeders ASAP (60-90 days)
Collect your own breeding statistics o Evaluate regularly
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Colony Management Tips
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Record and investigate deviations
o Environmental changes? o Breeding errors? (check genotypes)
Expect changes on a new background
o Keep previous generations while evaluating
Refresh colony every ~ 10 generations
o Replace breeders from a trusted vendor o Backcross to inbred or F1 hybrid parent
Maintain pedigreed colonies
o Filial (sister x brother) matings
www.jax.org/jaxmice/support/husbandry/room-conditions
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Maintaining a Pedigreed Colony
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Single Established Colony - any strain type sister-brother mating only!
Pedigree 1 Pedigree 2
F1
F2
F3
Prevent substrain development - refresh every ~10 generations
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Mating Options
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Pair: one female x one male
Trio: two females x one male (same cage) o “aunting” phenomenon
Harem: single male, more than two females o NOT recommended
Male rotation: two females x male (week 1), same male, two new females (week 2) o Single mutant male, need many offspring o Male has a very short lifespan (neuro. mutants)
www.jax.org/jaxmice/support/husbandry/colony-planning
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Tips for Poor Breeders
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Quiet place
Ensure adequate darkness Minimal handling Use clean forceps or gloves Change dietary fat content Add enrichment Leave mating pairs together
www.jax.org/jaxmice/support/husbandry/poor-breeders
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What do you do if your colony is breeding
poorly?
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How to Foster a Litter
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Select foster mother o Different coat color o Has successfully weaned a litter (ideal) o Has a near age matched litter (ideal) Remove natural litter and reduce in size
Combine foster and natural pups
Gently mingle pups with soiled shavings from foster cage
Place all pups in foster cage
Do not disturb
Pups gathered into the nest is a good sign www.jax.org/jaxmice/support/husbandry/fostering-litters
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Reducing Costs & Saving Space
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Size colony for your needs
Use both sexes or an age range
Consider ordering cohorts of mice
Rotate breeders on a strict schedule
Replace nonproductive breeders ASAP (60 – 90 days)
Run a tight ship…appoint a trusted colony manager o Maximizes efficiency Cryopreserve unique and low-use strains
www.jax.org/jaxservices/cryopreservation/calculator
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Cryopreservation
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For colony “sleep”
For insurance and peace of mind o Development and basic phenotyping of a genetically-modified
strain can take 2-3 years and cost more than $100,000
Can you afford not to preserve your strains?
Cryopreservation & Recovery Services |
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The Jackson Laboratory Patented Genetic Stability Program
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www.jax.org/jaxmice/genetichealth/stability
US patents 7592501, 8110721
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Choosing a Breeding Scheme
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What genotypes do I need? o How can I identify them?
What controls are available?
Linkage considerations o X-linked or autosomal?
Multiple genes o Linked or segregating independently?
Reproductive considerations (sterility or subfertility?)
Embryonic or postnatal lethality?
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Homozygous Breeding Scheme
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Background Controls inbred or congenic inbred* mixed background (e.g. B6;129) outcross to F1 every ~10 generations
F2 hybrid approximate control*
- - - -/- -/- - -/- -/-
Both genders viable and fertile as homozygotes
*Determine acceptable controls in your area of study Genotype new breeders for insurance
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Heterozygous Breeding Schemes
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Het (+/-) x Hom (-/-) 50% homozygous offspring
Het (+/-) x Het (+/-) 25% homozygous offspring
Background Controls inbred, congenic or mixed background wild-type or het sibling*
- - + +/- +/- - -/- -/-
+ - + +/+ +/- - +/- -/-
Genotyping typically required each generation
One (or both) genders not viable or fertile as homozygotes
* if no phenotype
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Considerations for Transgenic Mice: Founder Lines & Integration Sites
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Tg expression may affect viability or fertility Ex: B6CBA-Tg(HDexon1)62Gpb/3J (006494)
o Huntington’s disease model o Tremors, seizures by ~ 9-11 weeks o Females infertile & 50% males sterile o Limited breeding window (3-4 weeks)
Insertional effects/mutations Ex: B6.Cg-Hmga2pg-Tg40BCha/BmJ (002644)
o Globin transgene inserted into pygmy locus (Hmga2pg) o Heterozygotes phenotypically normal o Homozygotes smaller in size; infertile
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** Evaluate multiple founders!
Hom Het
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Initial Mating Scheme for Transgenic Mice
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Background Controls inbred or mixed background wild-type (noncarrier) sibling
Tg + + Tg/+ +/+ + Tg/+ +/+
Wild-type noncarrier (+/+) x Hemizygote (Tg/+ or Tg/0) 50% of offspring carriers
Are homozygous (Tg/Tg) viable? Undesirable phenotype?
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Complex Breeding Schemes
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No standard PCR assay for genotyping o e.g. B6;C3Fe a/a-Csf1op/J (000231)
Homozygous op/op o Small, deficient bone marrow o Low fertility, reduced viability
Heterozygous (op/+) & wild-type (+/+) o Phenotypically normal; indistinguishable
Breeding options: o Progeny testing o Ovarian transplant
Normal bone
op/op bone
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Complex Breeding Schemes: Ovarian Transplantation
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X
op/op
X
op/+ op/+
?/+ ?/+ ?/+
all obligate hets
op/op
transfer ½ ovary
+/+
incomplete ovariectomy
ovariectomized host
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Usefulness of Ovarian Transplantation
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Circumvent genotyping difficulties/costs
o Unknown or unmapped mutations
Poor female reproduction but gametes viable
o Mouse models of obesity and diabetes
Extend breeding span of severe phenotype strains
o Huntington’s disease models o Amyotrophic lateral sclerosis (ALS) models
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Complex Breeding Schemes: Cre-Lox
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“floxed” target gene
“knockout” allele
X G
eneX
LoxP
GeneX
LoxP “
LoxP GeneX
LoxP
Cre excision
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Complex Breeding Schemes: Cre-Lox
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x loxP mouse -
“floxed” gene/allele
50% heterozygous for Gene x conditional knockout after 1 generation
loxP GeneX loxP
GeneX loxP loxP
cre transgene with liver-specific expression
Ex: B6.Cg-Tg(Alb-cre)21Mgn/J
GeneX
loxP GeneX loxP
Alb cre
Alb cre
GeneX
GeneX
Cre-lox mouse
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Complex Breeding Schemes: Cre-Lox
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Alb cre
GeneX
loxP mouse - “floxed” gene/allele
x loxP GeneX loxP
GeneX loxP loxP
heterozygous Cre-lox mouse
loxP GeneX loxP
loxP GeneX loxP
loxP GeneX loxP
Alb cre
25% Homozygous for conditional knockout after 2 generations
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Complex Breeding Schemes: Cre-Lox
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Offspring: 50% heterozygous knockout after 1 generation
Breeding & Rederivation Services |
x loxP mouse -
floxed gene/allele
EIIa Cre
loxP GeneX loxP
loxP GeneX loxP
GeneX
GeneX
GeneX
Cre mouse – cre transgene (Tg) widespread expression promoter
FVB/N-Tg (EIIa-Cre)C5379Lmgd/J
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Complex Breeding Schemes: Cre-Lox
36 Breeding & Rederivation Services |
Offspring 2nd generation: 25% homozygous knockout
GeneX GeneX
X
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Summary
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Be aware of the breeding characteristics of your strains
Keep good records & evaluate them regularly
Choose most effective breeding strategy to meet research needs
Consider cost saving options
o Use both sexes, age range o Cryopreservation
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Thank you!
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In need of mouse breeding and colony management expertise to advance your research? Contact your regional representative today www.jax.org/jaxmice/support/regionalcontacts
Contact technical support www.jax.org/jaxmice/support/techsupport-index
JAX® Mice, Clinical & Research Services
1-800-422-6423 • 1-207-288-5845 [email protected] • www.jax.org/jaxmice