Setting up and breeding a GM mouse colony. Anne Heikkinen, PhD, University of Oulu

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1 Setting up and breeding a GM mouse colony Anne Heikkinen, PhD, University of Oulu

2 Outline of the presentation Generation of mouse models Backcrossing Maintaining colonies Breeding schemes Problems Optimization and back-ups

3 Techniques to generate mouse models TRANSGENIC ES CELL- DERIVED CRISPR/Cas9 Tg+ Tg+ Tg+ Zygotes from an outbred/inbread strain Genetic background - zygote Zygotes from a desired inbred strain Genetic background - zygote Morulas/blastocysts from a convinient strain Genetic background - ES cells Modified from Young et al. 2015

4 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 backcrossing for > 10 generations while maintaining heterozygosity 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 given mutation can be observed depending on the genetic background used Applies also to fertility Complete strain nomenclature includes full substrain information, for example B6.129-Col13a1 tm4.1pih Current background Original background Modified gene Modification type Lab code Modified from

5 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 the inbread strain achieved WT from an inbread strain (e.g. BL/6N) can be used as control N<6-10, the approppriate control is a littermate control Not to establish independent WT and mutant lines with mixed background at early backcrossing generation! A JAX Handbook - Genetically Standardized Mice

6 Speed congenic method Genotyping for genome-wide polymorphic markers Mice with highest inbread background markers selected for further breedings ~99.8% achieved in ~5 generations Fast but laborous BL/6J and BL/6N sub-strains have been separated for at least 220 generations 34 coding SNPs, 2 coding small indels 146 non-coding SNPs, 54 non-coding small indels 27 phenotypic features significantly different ophthalmology, cardiovascular health, metabolism, behavior, clinical chemistry, hematology and immune function A JAX Handbook - Genetically Standardized Mice

7 Maintaining mice stem and expansion colonies Stem To maintain the line Housed in barrier Optimal breeding productivity scheme Long generation intervals If inbred filial breedings If backcrossing at least N10 Expansion To produce mice for analysis Housed in an approppriate facility Optimal breeding scheme for analysis Limited number of generations Any male femal pairs

8 Maintaining stem colonies - 1) Continuous backcrossing Backcrossing carriers at each generation with wild-types from a standard inbred strain The recommended way to maintain a mutant colony Eventually all mice resemble the inbread strain except for the mutation and possible flanking allele region will gradually reduce in size Heterozygous animals to generate WT and KO littermates readily avalable Left-over heterozygous can be used for further maintanance with the inbread strain

9 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. BL/6N) or littermates Inbreeding exposes to accumulation of mutations = genetic drift A deleterious mutation may spread to the entire colony fast Novel phenotypes Decrease in fertility and litter size Backcrossing every 5-10 generations

10 Breeding expansion colonies - Heterozygous matings with maximum genetic variation Breeders are chosen Randomly Nonrandomly by refering animals that are not closely related Control animals littermates, especially when mixed genetic background Animals to be studied (+/+ and -/- groups) chosen from as many breeding pairs as possible Special care need to be taken when combining samples from very different backcrossing generation (genetic background varies)

11 Maintaining a small mutant colony by random breeding No separate stem and expansion colonies Breeders are chosen randomly/nonrandomly by refering animals that are not closely related Eventually leads to inbreeding due to a limited colony size Inbreeding exposes to accumulation of mutations = genetic drift A deleterious mutation may spread to the entire colony fast Novel phenotypes Decrease in fertility and litter size Backcrossing every 5-10 generations

12 Maintaining genetic quality An inbread strain used for backcrossing is also exposed to a genetic drift Frozen embryos are used to refresh the stock every 10 generations At any time, mice in the inbread strain are only up to 10, 11, 12 generations away Researcher 1 Researcher 2 F10 F10 Researcher 1 Researcher 2 Frozen stock Researcher 1 Researcher 2 F10 F10 Researcher 1 Researcher 2

13 Breeding schemes 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!

14 Possible adverse effects of homozygous breedings 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 Foster mother If suspecting a maternal effect Using heterozygous mothers and analysing littermates

15 Basics in laboratory mouse reproductivity Average BL/6J (JAX ) Fertility rate varies 50% Gestation 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 months A JAX resource manual Breeding strategies for maintaining colonies of laboratory mice Isogenic/congenic mutants resemble the inbread strain, if not: Phenotype/spontaneous phenotype

16 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 reproductivity declines (< 8 months) No litter within 60 days of breeding fi breeding with a young WT If a male, sperm can be frozen fi in vitro fertilization If a female fi ovary transplantation Backcrossing at least every 10 generations to prevent genetic drifting Hox! Avoiding selective pressure Not selecting breeders only from litters of best breeders

17 Optimizing breeding performance Mating mice as early as they gain sexual maturity Breeding in trios (1 male x 2 females) produces best results in general Pregnant mothers are individually caged In case maximum productivity over time is required Continuous breedings since mating usually occures shortly after delivery 1 male x 1 female In case a large number of same-aged pups is required Housing as many females as possible in the same cage to synchronice the estrus cycle prior to starting breedings

18 Back-ups Protection agains spontaneous phenotypes Maintaining several independent stem colonies an unwanted spontaneous mutation can be readily detected and it does not spread within the entire colony Protection agains decline in breeding performance Keeping breeding pairs from more than one generation Not euthanizing previous generation before the current generation has proved fertile CRYOPRESERVING THE LINE AS EARLY AS POSSIBLE!

19 References A Jackson Laboratory Handbook - Genetically Standardized Mice idea_breed_a_mickey_mouse.html A Jackson Laboratory resource manual Breeding strategies for maintaining colonies of laboratory mice Coleman J.L., Brennan K., Ngo T., Balaji P., Graham R.M., Smith N.J.. Rapid Knockout and Reporter Mouse Line Generation and Breeding Colony Establishment Using EUCOMM Conditional-Ready Embryonic Stem Cells: A Case Study. Front Endocrinol (Lausanne) :105. doi: /fendo ecollection Crusio W.E., Goldowitz D., Holmes A. and Wolfer D. Standards for the publication of mouse mutant studies. Genes, Brain and Behaviour, 2009, 8:1-4. Papaioannou V. and Behringer R.R. Mouse Phenotypes: A Handbook of Mutation Analysis. Cold Spring Harbour Laboratory Press, Cold Spring Harbour, New York, Brennan K. Colony Pease S. and Saunders T.L. (ed.) Advanced Protocols for Animal Transgenesis: 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 of genome editing to investigate male reproductive mechanisms using mouse models. Asian Journal of Andrology, 2015, 17: