Effective Colony Management Mouse 101 Lecture 9/21/15 Wendy du Bois, Biologist NCI/CCR/LCBG

Why Use Mice as a Model 1. Biologically very similar to humans: 95% shared genes, very similar immune systems, get similar diseases for many of the same genetic reasons 2. Can manipulate the genome directly and model specific human diseases 3. Inbred mice are available that are genetically identical to each other. Increases accuracy and reproducibility in experiments 4. Accelerated life span: 1 year in a mouse= 30 years in a human 5. Have been used in research for more than 100 years. Very well understood and described 6. Cost effective- small, reproduce quickly, easy to handle and transport

Mouse Facts Life span: 1-3 years Neonate= birth to wean (~21 days), begin eating solid food at 12-14 days Sexual maturity 5-8 weeks (5-7 for males) Adult size at 8-10 weeks Geriatric at 18+ months

Breeding Objectives Maximize pup yield to meet research goals Maximize quality- healthy offspring of the desired genotype Minimize cost- avoid excess pups (genotype early), recognize and replace poor breeders

Setting Up Breeder Pairs Sexual maturity 5-8 weeks- set up 6-8 weeks* Breeders should be replaced every 7-8 months (optimal breeding age 2-10 months) Replace breeders if they are not productive 1. no litter 60 days after mating (90 for mutant strains) 2. 60 days since last litter 3. 2-3 litters born with no weaned pups 4. can try rotating males Don’t set up too early can lead to cannibalized litters or distocia before retiring cage make sure there is a male and a female, not 2 females…

Mice can be set up as either: Monogamous pairs: one male with one female This takes advantage of the post partum estrous cycle and can reduce the chance of fighting Takes up space, so higher cost. Older litter has to be weaned before new litter is born. Can be a problem with difficult strains Harem Mating: 1 male with 2-3 females Pregnant females must be separated to prevent over crowding Can be difficult to track progeny Helpful if you need to produce larger numbers or if you have a limited number of males

1. Gestation 18.5-21 days 2. Litter size 2-12+ pups 3. Weaning age 17-28 days Standard weaning age is 21 days Try not to disturb the cage for 2-3 days before birth and 3-5 days after especially if it is a difficult strain Runted pups can be left in the breeder cage longer than 21 days, or can be supplemented with soft gel food When weaning, males can be combined. This is the only time you can combine males, otherwise they will fight

Be alert to changes in productivity Things that can influence breeding: 1. Hybrid vigor 2. Post natal effects 3. Parental behavior 4. Genetic background 5. Diet- high fat vs low fat If your pups are being cannibalized, things to consider: 1. Is it the mom’s 1st litter? 2. Is the male aggressive? 3. Is there a 2nd litter dba= poor breeder, balbc = average breeder, CDF1= excellent C57bl susceptible to hydrocephaly and micro opthalmia Sjl males are extremely aggressive and might cannibalize litters C57bl males are helpful with raising pups High fat can increase productivity, but if the mice become obese, can slow or stop productivity. Use a mix we give sunflower seeds

Fostering At Risk Litters Necessary if mother dies or is neglectful, has poor milk production (look for milk spot) or if her litter is too large. Use a mouse with a different coat color. Choose a female that has already successfully weaned a litter. Be sure she has a litter of pups that are roughly the same age Sac some of her pups if necessary Move the foster mom to a clean cage Using clean gloves add the pups to her dirty cage and mix the pups in with her litter Wait a few minutes, then return her to the cage. You can add a treat to distract her. Watch from afar- she should begin to care for the pups within one hour DO NOT forget to record what you have done on the cage card!!

Environment Is Crucial to a Productive Colony 1. Temperature: 68-79 degrees 2. Light cycle and intensity 3. Noise/vibration 4. Air pressure and humidity 5. Odors 6. Can be sensitive to changes in caretaker staff 7. Season 8. Enrichment Continuous lighting can halt breeding in 3 days Location on the shelf Moving equipment, near a cage wash, vibration attenuation pads Toxic fumes, cleaning chemicals perfumes (should be prohibited) Increase in breeding in spring and again in late summer Nesting material, chew toys, shacks.

Genetic Modification Things to consider: 1. May lead to embryonic lethality-common in homozygotes 2. Infertility 3. May lead to poor mammary function 4. Disease: tumor development, neurodegeration Still can be interesting because they can highlight the time and place the gene is first required during embryonic development Can genotype embryos using the egg sac by pcr May only be one sex- will affect how you set up your breeders If these issues arise at an early age, can def affect breeding.

Animal Health Issues Signs that your mouse is sick: 1. Scruffy coat 2. hunched, sunken at hips 3. weight loss 4. labored breathing 5. skin lesions 6. eye or nasal discharge 7. abnormal behavior Alone in cage

Beginning a New Colony 1. generally a good idea to start with 2-4 breeder pairs if possible. 2. how will you be identifying the mice you want to use? Genotype? PCR or Southern Phenotype? Breeding in the case of x-linked genes

If Genotyping Can use either small piece of tail or a piece of ear tissue Various kits available for extracting DNA Phenol Chloroform and other precipitation methods HotSHOT: Alkaline lysis reagent Heat at 95 degrees for 10minutes to and hour (30 minutes optimal) Cool to 4 Add TrisHCl neutralization buffer Use 1-3ul directly Store at 4 to -20 Great way to genotype egg sacs

Mouse Identification Neonates: Toe clip (up to 7 days) Ear notch Tattoo

Weanlings/Adults: Ear Notch: inexpensive, can use tissue for genotyping/can tear and heal Ear Tag: relatively inexpensive, unique numbering/ can fall out, infection, difficult to read, mice have to be older

Tattoo: permanent, easy to read/difficult to do, time consuming, can fade Microchips: permanent, unlimited numbers, can also record biometric information/expensive

Data Collection/Record Keeping Critical to animal studies Maintain a pedigree book or database Include: History of strain Name changes Generation number (N=number of times mated to the background strain, F= filial or number of times brother sister mated, p= cryopreserved) N6F4p Genotypes/tissue numbers Dates of mating Date cage retired Info about quality control tissues (what kind, location) Litter information Our pedigree records go back to 1972

Cage Cards Should include: Protocol number Investigator name Strain name DNA info (id number/genotype) Sex Number of mice in cage Family history for stock/wean cards and breeder cards

Breeder Cards Can get important information from breeder cards: Date female found pregnant Date litter born Litter size at birth Number of pups weaned Ratio of m:f at wean

Keep ALL Cards! Use different colors for different strains Can make specialized cards-timed pregs etc.

Mouse colony management software options Excel spreadsheets Filemaker JaxColonyManagementSystem (JCMS) http://colonymanagement.jax.org free, can print cards, manage experiments Softmouse https://www.softmouse.net Has a free version, depends on size of lab/colony Labguru www.labguru.com Can link with specimen storage info, $13.00/user/month Mousecolony www.mousecolony.com ~$550/user

Long Term Considerations Maintain founder stocks Check for Genetic Quality control Keep reference tissues at a set number of generations (ie every time you advance and N generation or every 5 F’s) Consider having strains tested by an outside source for “purity” Dartmouse ($149/sample) Jackson Labs ($159/sample) Charles River

Consider Cryopreservation Gets mice you are not actively using off the shelf- saves $$$$ Provides a way to eliminate pathogens Helps to prevent genetic drift/mutations Disaster prevention Insurance and peace of mind: Development and basic phenotyping of a typical strain is 2-3yrs and >$100,000

Methods of Cryopreservation Embryo: $312/strain, usually have to provide 4-6 males which are mated to comercially available inbred females Ovary: $644/strain, useful for strains with “weak” embryos Sperm: $990/strain, get 15 straws and 3yrs storage Jax also has a “do it yourself” sperm freezing kit- $3750 for 3 strains, includes shipping, QA and storage for 3 years.

Consider Donating Your Strain NCI mouse repository: funded by NCI for mouse cancer models. Strains made available to all members of the scientific community. Jackson Labs: you pay to ship, strains are rederived

On Line Resources Available 1. Jackson Labs Mouse Database: www.jax.org 2. Mouse genome informatics: www.informatics.jax.org (genome database, gene expression, tumor biology, polymorphisms) 3. Jax Phenome Database: www.phenome.jax.org (collaborative, standardized collection of measured data, includes baseline phenotype data) 4. International Mouse Strain Resource: www.findmice.org (searchable online database of mouse strains. Goal is to assist the international scientific community in locating and obtaining mouse resources for research)

Services Available at NIH NCI/Frederick: A. Colony expansion B. Speed congenics C. Generation of transgenic and gene targeted mice D. Small animal imaging (xray, ct, pet, optical bioluminescence and fluorescence) E. Pathology/help with study design F. Embryology G. Blood chemistry analysis/hematology Possible to get 500 offspring from a single male 10 backcrosses = 99% identical to inbred strain -2.5-3 years uses microsatellites (over 6000),many of which are polymorphic between different inbred strains, reduces the number of backcrosses to 5 and time to about 12-14 months

Others Division of Veterinary Resources (DVR) NHLBI Phenotyping Core 1. Bacteriology 2. diagnostic support 3. health surveillance/monitoring 4. nutrition 5. pharmacy 6. behaviorist 7. irradiation 8. pathology NHLBI Phenotyping Core 1. Cardiovascular phenotyping 2. metabolic phenotyping 3. neuromuscular 4. behavioral 5. exercise physiology 6. advanced imaging modalities Submit fecals…

The End! Thanks to Jackson Labs