Effective Colony Management

Mouse 101 Lecture9/21/15

Wendy du Bois, BiologistNCI/CCR/LCBG

Why Use Mice as a ModelO 1. Biologically very similar to humans: 95%

shared genes, very similar immune systems, get similar diseases for many of the same genetic reasons

O 2. Can manipulate the genome directly and model specific human diseases

O 3. Inbred mice are available that are genetically identical to each other. Increases accuracy and reproducibility in experiments

O 4. Accelerated life span: 1 year in a mouse= 30 years in a human

O 5. Have been used in research for more than 100 years. Very well understood and described

O 6. Cost effective- small, reproduce quickly, easy to handle and transport

Mouse FactsO Life span: 1-3 years

O Neonate= birth to wean (~21 days), begin eating solid food at 12-14 days

O Sexual maturity 5-8 weeks (5-7 for males)

O Adult size at 8-10 weeksO Geriatric at 18+ months

Breeding ObjectivesO Maximize pup yield to meet research

goals

O Maximize quality- healthy offspring of the desired genotype

O Minimize cost- avoid excess pups (genotype early), recognize and replace poor breeders

Setting Up Breeder Pairs

O Sexual maturity 5-8 weeks- set up 6-8 weeks*

O Breeders should be replaced every 7-8 months (optimal breeding age 2-10 months)

O Replace breeders if they are not productive

1. no litter 60 days after mating (90 for mutant strains)

2. 60 days since last litter3. 2-3 litters born with no weaned pups4. can try rotating males

Mice can be set up as either:

O Monogamous pairs: one male with one femaleO This takes advantage of the post partum estrous

cycle and can reduce the chance of fightingO Takes up space, so higher cost. Older litter has

to be weaned before new litter is born. Can be a problem with difficult strains

O Harem Mating: 1 male with 2-3 femalesO Pregnant females must be separated to prevent

over crowdingO Can be difficult to track progenyO Helpful if you need to produce larger numbers

or if you have a limited number of males

O 1. Gestation 18.5-21 daysO 2. Litter size 2-12+ pupsO 3. Weaning age 17-28 days

Be alert to changes in productivity

O Things that can influence breeding:O 1. Hybrid vigorO 2. Post natal effectsO 3. Parental behaviorO 4. Genetic backgroundO 5. Diet- high fat vs low fat

O If your pups are being cannibalized, things to consider:O 1. Is it the mom’s 1st litter?O 2. Is the male aggressive?O 3. Is there a 2nd litter

Fostering At Risk LittersO Necessary if mother dies or is neglectful,

has poor milk production (look for milk spot) or if her litter is too large.

1. Use a mouse with a different coat color.

2. Choose a female that has already successfully weaned a litter.

3. Be sure she has a litter of pups that are roughly the same age

4. Sac some of her pups if necessary

1. Move the foster mom to a clean cage

2. Using clean gloves add the pups to her dirty cage and mix the pups in with her litter

3. Wait a few minutes, then return her to the cage. You can add a treat to distract her.

4. Watch from afar- she should begin to care for the pups within one hour

5. DO NOT forget to record what you have done on the cage card!!

Environment Is Crucial to a Productive Colony

O 1. Temperature: 68-79 degreesO 2. Light cycle and intensityO 3. Noise/vibrationO 4. Air pressure and humidityO 5. OdorsO 6. Can be sensitive to changes in

caretaker staffO 7. SeasonO 8. Enrichment

Genetic ModificationO Things to consider:

O 1. May lead to embryonic lethality-common in homozygotes

O 2. InfertilityO 3. May lead to poor mammary

functionO 4. Disease: tumor development,

neurodegeration

Animal Health IssuesO Signs that your mouse is sick:

O 1. Scruffy coatO 2. hunched, sunken at hipsO 3. weight lossO 4. labored breathingO 5. skin lesionsO 6. eye or nasal dischargeO 7. abnormal behavior

Beginning a New Colony

O 1. generally a good idea to start with 2-4 breeder pairs if possible.

O 2. how will you be identifying the mice you want to use?

O Genotype? PCR or SouthernO Phenotype? O Breeding in the case of x-linked genes

If GenotypingO Can use either small piece of tail or a piece

of ear tissueO Various kits available for extracting DNAO Phenol Chloroform and other precipitation

methodsO HotSHOT:

O Alkaline lysis reagentO Heat at 95 degrees for 10minutes to and hour

(30 minutes optimal)O Cool to 4O Add TrisHCl neutralization bufferO Use 1-3ul directlyO Store at 4 to -20

Mouse IdentificationO Neonates:

O Toe clip (up to 7 days)

O Ear notchO 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

O Tattoo: permanent, easy to read/difficult to do, time consuming, can fade

O Microchips: permanent, unlimited numbers, can also record biometric information/expensive

Data Collection/Record KeepingO Critical to animal studiesO Maintain a pedigree book or

databaseO Include: History of strain

Name changesGeneration number (N=number of times mated to the background strain, F= filial or number of times brother sister mated, p= cryopreserved)

N6F4pGenotypes/tissue numbersDates of matingDate cage retiredInfo about quality control tissues (what kind, location)Litter information

Cage CardsO Should

include: O Protocol

numberO Investigator

nameO Strain nameO DNA info (id

number/genotype)

O SexO Number of

mice in cage

Breeder CardsO Can get important information

from breeder cards:O Date female found pregnantO Date litter bornO Litter size at birthO Number of pups weanedO Ratio of m:f at wean

O Keep ALL Cards!

O Use different colors for different strains

O Can make specialized cards-timed pregs etc.

Mouse colony management software options

O Excel spreadsheetsO FilemakerO JaxColonyManagementSystem

(JCMS)http://colonymanagement.jax.org

free, can print cards, manage experimentsO Softmousehttps://www.softmouse.netHas a free version, depends on size of lab/colony

O Labguruwww.labguru.comCan link with specimen storage info, $13.00/user/month

O Mousecolonywww.mousecolony.com

~$550/user

Long Term Considerations

O Maintain founder stocksO Check for Genetic Quality control

O Keep reference tissues at a set number of generations (ie every time you advance and N generation or every 5 F’s)

O Consider having strains tested by an outside source for “purity”O Dartmouse ($149/sample)O Jackson Labs ($159/sample)O Charles River

Consider CryopreservationO Gets mice you are not actively using

off the shelf- saves $$$$

O Provides a way to eliminate pathogensO Helps to prevent genetic drift/mutationsO Disaster preventionO Insurance and peace of mind:

O Development and basic phenotyping of a typical strain is 2-3yrs and >$100,000

Methods of Cryopreservation

O Embryo: $312/strain, usually have to provide 4-6 males which are mated to comercially available inbred females

O Ovary: $644/strain, useful for strains with “weak” embryos

O Sperm: $990/strain, get 15 straws and 3yrs storage O 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

O NCI mouse repository: funded by NCI for mouse cancer models. Strains made available to all members of the scientific community.

O Jackson Labs: you pay to ship, strains are rederived

On Line Resources Available

O 1. Jackson Labs Mouse Database: www.jax.org

O 2. Mouse genome informatics:www.informatics.jax.org (genome

database, gene expression, tumor biology, polymorphisms)

O 3. Jax Phenome Database: www.phenome.jax.org (collaborative,

standardized collection of measured data, includes baseline phenotype data)

O 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

O NCI/Frederick: O A. Colony expansionO B. Speed congenicsO C. Generation of transgenic and gene

targeted miceO D. Small animal imaging (xray, ct, pet,

optical bioluminescence and fluorescence)O E. Pathology/help with study designO F. EmbryologyO G. Blood chemistry analysis/hematology

OthersO Division of Veterinary Resources

(DVR)O 1. BacteriologyO 2. diagnostic supportO 3. health surveillance/monitoringO 4. nutritionO 5. pharmacyO 6. behavioristO 7. irradiationO 8. pathology

O NHLBI Phenotyping CoreO 1. Cardiovascular phenotypingO 2. metabolic phenotypingO 3. neuromuscularO 4. behavioralO 5. exercise physiologyO 6. advanced imaging modalities

The End!

Thanks to Jackson Labs