Mouse Care

4/25/06

1. Mice are housed in GSRBII room 2027. This is a barrier facility of the cleanest level. You cannot enter the facility is you have visited another animal facility on the same day. So if you need to go to the animals in Bryan, do that later in the day after GSRBII.

2. Mice need to be attended biweekly, best days are Tuesday and Friday since the cages in our room are changed out on Wednesday and Thursday by DLAR staff, meaning that people will be in the room with the hood taking up most of the space.

3. Before leaving for GSRB, print out the table of animals for each colony to be tended from the Filemaker Pro files on the USB key attached to the mouse notebook. All lab computers have FilemakerPro, so you can open the file on any computer, but please save the file to the key so everyone can access it. The logs will list each cage as

“cross” meaning one male and one female are in the cage mating

“multicross” meaning one male with more than one female are mating

“pregnant” meaning there is a pregnant female before birth or up to weaning

“weaned” meaning these are mice more than 21 days old, but not yet mated

“separated male/female” - a mouse that has mated before but isn’t crossed now.

4. Take with you to the animal facility – the black notebook labeled “GSRBII animals”, a ball point pen, and the “Tail” box, which should contain small sharp scissors, many (>20) eppendorf tubes filled with 300uL 70% ethanol, and a black Sharpie for tube labeling.

5. Enter through the dressing room. Dress required to enter the facility is body suit, hair cover, face mask, gloves, and booties. Put on the booties last foot by foot as you step over the bench to enter the facility.

6. If the hoods are in the hall, bring one in with you, turn it on, and clean it off. You cannot open any cages in the room unless they are in a hood. If there are no hoods in the hall, then they are both in use. You can bring the cages to the procedure room (first room on the left within our module), where they can be opened on the benches.

7. Look in on each cage and first scan the cards. If any messages have been left by DLAR staff (especially “overcrowded”) you will need to respond to their needs right away. If any temporary cards on in place, make sure to look that the printed cards have been left on the table in the room and place them on the correct cages.

ABOUT OVERCROWDING: Each cage can contain up to 5 adult mice, or up to two adult mice with one litter of pups up to the age of weaning (21 days of age). If weanlings are sick and need to be kept with the mother longer, you can put a “special husbandry conditions” card on the cage and ask to delay the date of weaning one week. It is important that the animals be weaned by the time the females are 4 weeks old, or else they become competent to breed.

8. Consult the table of animals for each colony and look at the notes to determine what to do with each cage.

“Multicross” cages are the most crucial because if more than one female were to become pregnant and litter in the cage, these would be overcrowded and we get charged to separate them. When the cross is set, mark in the notes “check for preg on [date]”. The date you write down is 5 visit days (about 18 days) after the day the cross is set. (ex: set the cross on Friday April 7^th. You write “check for preg on Tuesday April 25^th”. Mice have a 19 day gestation and a 4 day ovulation cycle. So once crossed, good breeding mice will generally give birth 20-24 days later. So checking 18 days after setting the cross will allow you to find the pregnant mice before the litters are born.

Pick up the females by the tail and look for big bellies to find the pregnant ones. If the females do not look pregnant, change the notes to read “check for preg” with the next date that you will come visit “4/28” for the example above. You then check the cage every visit. If a female is pregnant in a multi-cross cage, move her to a new cage. One female can be left with the male, and change the designation of the cage to “cross”. It is ok to have two adults with one litter in a cage.

ABOUT MAKING NEW CAGES: Caging materials are in the hall. Make sure each new cage has the lixit water dispenser in the back, a top, a food pellet dispenser filled with food (food is in a bin in the room), and a card holder. When you make a new cage, put a temporary card in the holder and make sure to note that you need to request a new card. The card request form is under “forms” as a typeable PDF on the USB key in the mouse notebook. Fill this out and FAX to the number on the form as soon as you return from the animal room so as not to forget. Make sure to fill in the form properly – you request cards in separate places for separating animals (because these have already been counted on the protocol) versus weaning animals (because these numbers need to be added to the protocol). Cards will be delivered the next day in the afternoon to our mouse room. On your next visit replace the temporary cards with these cards. Remember, we can only put animals into our designated spaces. Do not put animals into cage space reserved for other labs.

“Cross” cages get treated essentially the same as the multi-cross cages described above. You do not need to remove the pregnant females from the male. If a female is found pregnant, change the designation of the cage to “Pregnant” and date it for your next visit as “Check for pups.” You will want to put a “Special Husbandry Conditions” card on the cage BEHIND the regular cage card. Check “caging” and date the card from the day you place it until one week from that date. At the bottom of the card write “pregnant mouse, PI will change cage”. That way the newborn pups won’t be disturbed on cage change day.

“Pregnant” cages will have one of two designations in the notes. If the pups were not yet born on your last visit, it will say “check for pups.” You want to just pull the cage out slightly from the rack and look underneath the mom to see if you see the pups. Try to disturb her as little as possible. If you see pups, estimate the approximate age. If they are still pink they are probably P0 or P1. If they have dark coloring appearing, they are closer to P3 or P4. Once you see pups and estimate their age, write in the note “ear clip and tail on XX” choosing the date of your visit when the animals will be closest to 10 days of age.

For pregnant cages that already had pups you will need to ear clip and tail the animals at 10 days of age. First get a new cage and move mom (and dad) over to get them out of the way. The ear clip diagram is in the mouse notebook. It will allow you to label up to 9 animals in one cage. If you have more than 9 animals, separate the males and females first and number them each 1-9. To tell males from females, lift them by the tail and look at their undersides. Females usually have obvious nipples up the sides of their bodies that the males lack. Females also have a shorter anus-genital distance than the males do, and the males’ genitalia look a little bigger than the females’. I write on the table of animals sheet in the black notebook 1-X for the number of pups, lift one, say if it is male or female, clip about 3mM of tail into an Eppendorf tube with ethanol, then clip the ears with the correct pattern for that animal number, and drop the pup into the new cage. Before moving on, I make sure the tail biopsy is in the tube, I close it, and I label with the number of the animal and the last three digits of the cage (1-063, for ex.)

Once the pups are tailed, you write in the notes “wean pups on XX” and choose the date of your visit when the pups would be at least 21 days old. On weaning day, you will need to separate the pups from the mom (and dad), sorting the males and females into different cages. If you are not sure of the sex, NOTE THIS on the animal table and recheck on a later visit. The #1 cause of overcrowded cages is poor sexing at the time of weaning resulting in unexpected litters from the weaned animals since these cages aren’t being watched as closely as the cross cages. Based on the genotypes you will probably only keep some of the animals, which you will identify by their ear clips (see why you need to do the clipping well?). You can keep up to 5 weanlings in one cage together. Sort the animals you won’t keep into one of the old dirty cages, as these animals will be euthanized (see below). Once the pups are weaned they get the designation “weaned” and the parents can go back into a cross or be pulled from crossing into holding cages designated “Separated male/female” to designate that they have mated before.

EUTHANIZING MICE: We sacrifice mice with CO2. The CO2 chamber is near the sink in the hall outside the animal room. Instructions for CO2 euthanasia are written on top of the plexiglas box. Place the mice to be euthanized inside and turn on the CO2 at the tank. After about 2-3 minutes, the mice should not be moving. Turn off the gas and leave mice inside for another 2 minutes. Mice are then removed and use the scissors on the side of the box to cut into both sides of the chest so the animal could not breathe if it happened to wake up and try to take a breath. Wrap up the mouse, put in a plastic bag, label the bad “west” and place in the dead animals box. If you have eliminated a cage, take the card off the cage, note the sacrifice date on the card and place in the card box which is on the same table as the CO2 chamber.

“Weaned” cages hold males and females of known genotype for experiments or future crosses. The only concern about these cages is that bad sexing can result in overcrowding, so look in on these cages 3-5 weeks after making them to ensure there are no unwanted litters coming. Females become competent to mates at 4 weeks (28 days), males at 6 weeks (42 days). Females will mate generally well up to 6 months of age, males up to 1 year of age. The table of animals tells the age of each animal if you enter the birthdate.

“Separated male/female” cages hold older animals that have successfully mated. They require little attention except an occasional look to make sure the animals are healthy.

9. Once you finish will all the cage work, make sure all cages are back in the rack and clicked tightly in so the water will flow (listen for the “click” as you push on the cage). Clean the hood and return it to the hall. Take all dirty cages to the cart out the back door of the module. Leave via the back door. You cannot reenter the module unless you go back through the dressing room and redress.

10. When you return to the lab, first make sure to fill out the cage card request form for any new cages you created. FAX this form to the number at the top.

11. Next, enter all of the changes that you made into the FileMaker Pro files for these animals. Start FileMaker on any computer open the files off the USB key. Choose the “Table of animals view” and update each cage. If you have weaned animals, you will need to create a new animal card for each one. Change the view to individual animal. Type control N to make a new record and type in the required information. The numbering for each animal should be the ear clip number (1-9), then the last three digits of the cage the animal came from, then the day/month date in 00/00 form without the slash. So animal 8 from cage 271049 born on 01/05/06 would have the number 80490105. Hopefully this system will keep the animals from having overlapping numbers. Go back to the table of animals view and sort the list by pressing control S. The files will automatically save back onto the USB key.

12. Once the data has been entered, make sure to lyse any tails collected for genotyping. Remove the ethanol and place tails in 300uL lysis buffer plus 2uL proteinaseK. Leave at 55 degrees overnight and ask someone else to pull the tails the next day if needed.