Setting up and breeding aGM mouse colony

Anne Heikkinen, PhD, University of Oulu

Outline of the presentation

• Generation of mouse models

• Backcrossing

• Maintaining colonies

• Breeding schemes

• Problems

• Optimization and back-ups

Techniques to generate mouse models

Modified from Young et al. 2015

Tg+ Tg+ Tg+

TRANSGENICES CELL-DERIVED CRISPR/Cas9

Morulas/blastocysts from a convinient strainGenetic background - ES cells

Zygotes from an outbred/inbread strainGenetic background - zygote

Zygotes from a desired inbred strainGenetic background - zygote

Terminology• Co-isogenic strain = differs from an inbred

strain by a mutation at one locus• For example CRISPR/Cas9-derived strains

• Congenic strain = created by backcrossingfor > 10 generations while maintainingheterozygosity at a selected locus(flanking alleles)

• Targeted, ES cell-derived mouse strains• Backcrossings of co-isogenics to a different

strain

• Widely divergent effects of a givenmutation can be observed depending onthe genetic background used

• Applies also to fertility

Þ Complete strain nomenclature includesfull substrain information, for example

B6.129-Col13a1tm4.1Pih

Modified from https://commons.wikimedia.org/wiki/File:Backcrossing.png

Currentbackground

Originalbackground

Modifiedgene

Modificationtype

Labcode

Generation of a congenic backgroung

• ∼10 generations (N), about 2,5 years• Gender changed at least once

• Mitochondrial DNA• Y chromosome

• Flanking alleles remain

• N>10, genetic uniformity with theinbread strain achieved

• WT from an inbread strain (e.g.C57BL/6N) can be used as control

• N<6-10, the approppriate controlis a littermate control

• Not to establish independent WT andmutant lines with mixed backgroundat early backcrossing generation!

A JAX Handbook - Genetically Standardized Mice

Speed congenic method• Genotyping for genome-wide

polymorphic markers• Mice with highest inbread back-

ground markers selected for furtherbreedings

• ~99.8% achieved in ~5 generations• Fast but laborous

• C57BL/6J and C57BL/6N sub-strainshave been separated for at least 220generations

• 34 coding SNPs, 2 coding small indels• 146 non-coding SNPs, 54 non-coding

small indels

• 27 phenotypic features significantlydifferent

• ophthalmology, cardiovascular health,metabolism, behavior, clinical chemistry,hematology and immune function https://commons.wikimedia.org/wiki/File:Dna-SNP.svg

A JAX Handbook - Genetically Standardized Mice

Maintaining mice –stem and expansion colonies

Stem Expansion

To maintain the line To produce mice for analysis

Housed in barrier Housed in an approppriatefacility

Optimal breeding productivityscheme

Optimal breeding scheme foranalysis

Long generation intervals Limited number of generations

If inbred – filial breedings Any male – femal pairs

If backcrossing – at least N10

Maintaining stem colonies -1) Continuous backcrossing

• Backcrossing carriers at each generation withwild-types from a standard inbred strain

• The recommended way to maintain a mutantcolony

• Eventually all mice resemble the inbread strainexcept for the mutation and possible flankingallele region

Þ will gradually reduce in size

• Heterozygous animals to generate WT and KOlittermates readily avalable

• Left-over heterozygous can be used for furthermaintanance with the inbread strain

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Maintaining stem colonies -2) Inbreeding

• For isogenic/congenic (>N10) lines

• Filial (sister x brother or parent x offspring) intercross (F)• F10 about 2,5 years

• All mice resemble each other

• Controls can be from the parietal recipient strain (e.g.C57BL/6N) or littermates

• Inbreeding exposes to accumulation of mutations =genetic drift

• A deleterious mutation may spread to the entire colonyfast

Þ Novel phenotypesÞ Decrease in fertility and litter size

• Backcrossing every 5-10 generations

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Breeding expansion colonies -Heterozygous matings with maximumgenetic variation• Breeders are chosen

• Randomly• Nonrandomly by refering animals that are not closely

related

• Control animals littermates, especially whenmixed genetic background

• Animals to be studied (+/+ and -/- groups) chosenfrom as many breeding pairs as possible

• Special care need to be taken when combiningsamples from very different backcrossinggeneration (genetic background varies)

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Maintaining a small mutant colonyby random breeding

• No separate stem and expansion colonies

• Breeders are chosen randomly/nonrandomly byrefering animals that are not closely related

• Eventually leads to inbreeding due to a limitedcolony size

• Inbreeding exposes to accumulation of mutations =genetic drift

• A deleterious mutation may spread to the entirecolony fast

Þ Novel phenotypesÞ Decrease in fertility and litter size

• Backcrossing every 5-10 generations

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Maintaining genetic quality

• An inbread strain used for backcrossingis also exposed to a genetic drift

• Frozen embryos are used to refresh thestock every ∼10 generations

Þ At any time, mice in the inbread strainare only up to ∼10, 11, 12… generationsaway

Frozenstock

F10

F10F10

F10

Researcher 1

Researcher 2

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Researcher 1Researcher 2

Researcher 1Researcher 2

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Breeding schemes

https://www.jax.org/news-and-insights/jax-blog/2011/september/cre-lox-breeding-for-dummies

https://www.practicallyscience.com/category/bio/animals/

• Simple breeding schemes• -/- x -/-• -/- x +/-• +/- x +/-• +/- x +/+• +/+ x +/+

• Complex breeding schemes• Hybrid srains with two or more mutant alleles• Cre and loxP strains

• Breeding scheme depends on a situation

• Keeping breeding records!

Possible adverse effects of homozygousbreedings• Mother homozygous – maternal environment different from WT• Fertility

• Lag in getting pregnant• Litter size

• Pregnancy – fetal development• Environment, energy• Small circulating factors penetrating placenta

• Delivery• Survival rate

• Nursing• Behavioral problems

• Feeding• Defects in milk composition/production

• If suspecting a maternal effectÞ Using heterozygous mothers and analysing littermates

Foster mother

Basics in laboratory mouse reproductivity

Average C57BL/6J (JAX®)Fertility rate varies 50%

Gestation 18 – 21 days 18.5 days

Litter size 2 - 12 pups 5.5 pups

Weaning age 3 – 4 weeks 4 weeks

Sexual maturity 6 – 8 weeks

Best age to start breedings 6 – 8 weeks

Generation time 3 months

Productive breeding time 7 – 8 monthsA JAX resource manual – Breeding strategies for maintaining colonies of laboratory mice

• Isogenic/congenic mutants resemble the inbread strain, if not:• Phenotype/spontaneous phenotype

Managing small colonies

• Maintaining minimum of ∼4-6 breeding pairs

• Maintaining mixed-aged breeding population• Pairs from different generation• Not eliminating one until the next produces• Especially important when backcrossing to a new strain

• Replacing breeders before their reproductivitydeclines (< 8 months)

• No litter within 60 days of breeding ® breeding with ayoung WT

• If a male, sperm can be frozen ® in vitro fertilization• If a female ® ovary transplantation

• Backcrossing at least every 10 generations toprevent genetic drifting

• Hox! Avoiding selective pressure• Not selecting breeders only from litters of best breeders

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Optimizing breeding performance

• In case maximum productivity over timeis required

• Continuous breedings since mating usuallyoccures shortly after delivery

Þ 1 male x 1 female

https://www.practicallyscience.com/category/bio/animals/

• In case a large number ofsame-aged pups is required

• Housing as many females aspossible in the same cage tosynchronice the estrus cycleprior to starting breedings

• Mating mice as early as they gain sexual maturity

• Breeding in trios (1 male x 2 females) produces best results in generalÞ Pregnant mothers are individually caged

Back-ups

• Protection agains spontaneous phenotypes• Maintaining several independent stem colonies

• an unwanted spontaneous mutation can be readilydetected and it does not spread within the entire colony

• Protection agains decline in breedingperformance

• Keeping breeding pairs from more than onegeneration

• Not euthanizing previous generation before thecurrent generation has proved fertile

• CRYOPRESERVING THE LINE AS EARLY ASPOSSIBLE!

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References

• A Jackson Laboratory Handbook - Genetically Standardized Mice

• A Jackson Laboratory resource manual – Breeding strategies for maintaining colonies oflaboratory mice

• Coleman J.L., Brennan K., Ngo T., Balaji P., Graham R.M., Smith N.J.. Rapid Knockout andReporter Mouse Line Generation and Breeding Colony Establishment Using EUCOMMConditional-Ready Embryonic Stem Cells: A Case Study. Front Endocrinol (Lausanne).2015 6:105. doi: 10.3389/fendo.2015.00105. eCollection 2015.

• Crusio W.E., Goldowitz D., Holmes A. and Wolfer D. Standards for the publication ofmouse mutant studies. Genes, Brain and Behaviour, 2009, 8:1-4.

• Papaioannou V. and Behringer R.R. Mouse Phenotypes: A Handbook of MutationAnalysis. Cold Spring Harbour Laboratory Press, Cold Spring Harbour, New York, 2005.

• Brennan K. Colony Pease S. and Saunders T.L. (ed.) Advanced Protocols for AnimalTransgenesis: An ISTT Manual. Springer, Verlag, Berlin, Heidelberg, 2011

• Young S.A.M., Aitken J.R. and Ikawa M. Advantages of using the CRISPR/Cas9 system ofgenome editing to investigate male reproductive mechanisms using mouse models. AsianJournal of Andrology, 2015, 17:623-627.

http://www.ironicsans.com/2007/08/idea_breed_a_mickey_mouse.html