SOP Rederivation-Colony Expansion

1. PLAN AHEAD. If you do not have the necessary numbers of mice of the correct sex and age (see SOPs for Rederivation by IVF or Natural Matings), you MUST expand your colony. This takes 10 weeks minimum (see Note 1 below). If you do not have the males and females for steps 2 and 3 below, this extends to 5 months (20 weeks)!

2. House 5 males (2 - 8 mo of age) at 1 male/cage for 2 weeks. If your strain is a poor breeder or not all progeny will be of the correct genotype, set up more cages.

3. House 10 or more females (4-8 wks of age) at 5 females/cage. You need 2 females per male.

a. House together for 2 weeks. It is important to keep the females at a high cage density. Goal is to synchronize the estrous cycle so they all give birth at the same time (see Note 2).

b. 10 females should be sufficient (see Note 3) but more females may be required if your strain is a poor breeder or you must genotype. It is best to have more females than less but this is balanced with cage costs.

4. After 2 weeks, add to new cage: 2 females + 1 male + nestlet + paper tube.

a. Obtain high fat breeder chow from caretaker. Do not give to mice yet.

b. Check for pregnancy after 2 weeks.

c. Remove any pregnant females and house at 2 pregnant females per (1) clean cage. Add paper tube + nestlet breeder chow.

d. Replace females with females from nonproductive cages. No male should be without a female unless all females are pregnant. Record on the cage card which males are good studs.

f. On cages with pregnant female(s), place a pink special request card stating: DO NOT DISTURB - LAB WILL CHANGE CAGE.

5. Check pregnant female cages daily and change the cage if dirty. If P0 to P3 pups are present do NOT disturb the cage.

a. Check cages by gently looking from the side or bottom. Do NOT open the lid. If newborns are seen, do not disturb until P4 (ears should be in normal position).

b. At P4, move the litter to a clean cage along with some of the nesting material. Remove the DO NOT DISTURB sign unless the other female is still pregnant.

c. If the litter size is equal or more than 10 pups and you can identify the males with certainty, cull the males. Alternatively separate the mothers + litters into individual cages.

d. If the combined litter for 2 females is small (< 7 pups total), place a pink special request card stating: POOR BREEDERS. BOTH FEMALES NEEDED FOR LACTATION. The pink special request card is important.

e. Sex pups at P10 (if not yet done) and cull excess males to give female pups a growth advantage. Never cull to < 4 pups unless you plan to foster the pups to another lactating female to save on cage costs.

f. At P14, contact Transgenic Core to confirm rederivation date.

6. Having sufficient females of the correct age is the limiting step in rederivation. Therefore it is important to continue breeding your mice to obtain an additional 10 - 14 females in the event that the rederivation fails or results in low # of pups. This allows the Transgenic Facility to quickly repeat the rederivation.

NOTES: 1. (2) weeks of isolation of 5 males and 10 females + (1) wk mating + (3) wks gestation (19-20 d) + (4) wks (weaning + 1 wk) = 10 wks.

2. Lee-Boot Effect : crowding 5 females / cage -- estrous cycle stops. Whitten Effect - females re-enter estrus and become receptive simultaneously when placed with a male → all mate and become pregnant at about the same time and pups born at about the same time.

3. If only 4 of 10 females get pregnant: 4 litters x 5 pups/litter = 20 pups, ~10 females + 10 males.