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

2. 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

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

4. 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

5. 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)
days since last litter
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…

6. 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

7. 1. Gestation 18.5-21 days 2. Litter size 2-12+ pups
3. Weaning age 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

8. 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

9. 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!!

10. Environment Is Crucial to a Productive Colony
1. Temperature: 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.

11. 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.

12. 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

13. 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

14. 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

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

16. 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

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

18. 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

19. 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

20. 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

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

22. Mouse colony management software options
Excel spreadsheets
Filemaker
JaxColonyManagementSystem (JCMS)
free, can print cards, manage experiments
Softmouse
Has a free version, depends on size of lab/colony
Labguru
Can link with specimen storage info, $13.00/user/month
Mousecolony
~$550/user

23. 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

24. 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

25. 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.

26. 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

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

28. 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 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 months

29. 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…

30. The End!
Thanks to
Jackson Labs