General Rules

The Transgenic and Gene Targeting Facility services the entire research community of Albert Einstein College of Medicine and routinely provides transgenic mice to many labs. Because of this the facility follows strict guidelines to maintain pathogen-free facility labs, offices and animal room. All applicants and parties seeking services from the facility must agree to the following conditions as a pretext to receiving services.

NO ONE IS ALLOWED TO ENTER THE FACILITY MAIN LABS AT PRICE ROOM B110 IF THEY HAVE BEEN IN CONTACT WITH ANY ANIMALS WITHIN THE PAST 24 HOURS.

NO ONE UNDER ANY CIRCUMSTANCE IS ALLOWED TO ENTER FACILITY ANIMAL ROOM SAVE FOR DIRECT FACILITY PERSONNEL. NO ANIMALS FROM ANY OTHER ANIMAL ROOM ARE ALLOWED TO BE HOUSED IN FACILITY ANIMAL ROOM.

LAB PERSONNEL WHO ARE BRINGING ANIMALS FOR A DISCUSSED AND AGREED UPON PROCEDURE MUST BRING ANIMALS DIRECTLY TO PRICE ROOM B110. THE LAB MEMBER BRINGING THE CAGE OF ANIMALS MUST NOT HAVE HAD ANY CONTACT WITH THE ANIMALS DIRECTLY AND MUST NOT ENTER ANY OTHER ROOM ON THE FLOOR BESIDES CHANIN 638A.

back to top

Individual investigators are responsible for the super ovulation and mating of candidate mice for any relevant procedure. Once a target date has been agreed upon, investigators must obtain super ovulation hormones from the facility. If a procedure has been agreed to, then the cost of the hormones is free. If an investigator needs the hormones and will not seek additional services from the facility, a small charge is incurred from the distribution of hormones. The super ovulation hormones used are Pregnant Mare Serum (PMS) and Human Chorionic Gonadotropin (HCG). PMS is distributed in the form of a frozen 50 IU microcentrifuge tube. HCG is distributed in the form of a 50 IU lyophilized powder in a microcentrifuge tube. Store both in -70° Celsius. Hormone administration must follow a strict schedule as illustrated. Keep in mind when scheduling the injections that you must give the first injection (PMS) three days before the agreed upon target date for the relevant procedure.

Inject PMS anytime between 2:00 PM to 5:00 PM as an IP injection, 0.1cc per mouse.

Dissolve the contents of the HCG tube into 1000µl of autoclaved distilled water. Inject HCG 46 hours after PMS injection, IP injection 0.1cc per mouse.

Immediately after HCG injection, set up your mating (if necessary), preferably a single cage with one hormonally treated female with one male. No more than two females per male is recommended. The next morning check to see if any females are plugged and separate any unplugged females. If any super ovulated female is not plugged at this point, they will not be able to plug for at least 5-7 days, therefore it is advised that they be removed and set aside to recover if they need to be used again.

back to top

Applicants must contact facility director and discuss his/her construct and expected phenotypes. Investigators must complete and submit an official Pronuclear Injection Form and a Health and Safety Review of Transgenic Mouse Constructs Form. Key permissions that must be obtained are:

AECOM Institute of Animal Studies committee protocol title and approval number for the relevant project.
AECOM Environmental Health and Safety Department approval date.

Once all permissions are obtained, investigators will submit 100 µg of DNA from an Endofree-Qiagen preparation. Investigators then cut the DNA with the appropriate enzyme, use small amount of DNA to verify complete cutting, and then run on a large gel (we prefer TAE SeaKem GTG agarose gel, from FMC Bioproducts catalog no. 50074). When you are checking the large gel use long wave UV light (short wave UV will damage DNA). Then cut out the gel slice containing the DNA insert. The facility will then purify the DNA. Standard pronuclear injections use FVB embryos by default unless another strain is specifically requested/discussed. Injection scheduling is determined on a first come, first served basis unless extraordinary circumstances exist.

19-20 days following injection, pups will be born and they will be kept in facility housing for an additional 14 days, then they will be transferred into the investigator's animal room through Institute of Animal Studies. Total procedure may take up to 2 months, we guarantee either 3 founders or 60 pups whichever comes first. Southern results must be submitted within 4 weeks after receiving animals before any discussion about re-injection, greater than 4 weeks results in full cost of re-injection.

back to top

ES Cell Injection

Applicants must contact the Gene Targeting Facility manager and discuss arrangements for ES cell injection. Because of fluctuations of demand, there can be a waiting list of 2 weeks to several months. Scheduling is on a first come first served basis. Investigators must fill out a ES Cell Injection Form as well as a Health and Safety Review for ES-cell Injections Form. Key information that individual investigators are responsible for are:

AECOM Institute of Animal Studies committee protocol title and approval number for the relevant project.

AECOM Environmental Health and Safety Department approval date.

Once all permissions are obtained, investigators will be given a 4 day week to inject their desired cell lines at a per diem cost. Investigators can choose to either inject the same cell line all four days or different lines each day. It is advised that in order to increase the chance of chimeras that a line be injected at least for two days. If investigators need advice in cultivating their cell lines, they can be directed to various experienced labs.

The facility is responsible for generating C57 blastocysts for each day of injection, the ES cell injection, and then the transfer of injected blastocysts into CD1 pseudopregnants. The facility will then house the transfers until they give birth, and then will call the respective lab to pick up their chimeras.

back to top

Lentiviral Transgenesis

Investigators must contact the facility and make special arrangements for lentiviral injection. The Investigator is responsible for making the lentiviral construct, the infection and purification of the lentivirus.

Vasectomy

Investigators needing mice vasectomized can contact the facility and make arrangements for the procedure. Investigators must fill out the Transgenic Miscellaneous Service Form. Investigators can choose to perform the procedure on mice currently residing in their own respective animal rooms or choose to order mice through Institute of Animal Studies and have the mice delivered to the facility animal room, but all arrangements in detail must be agreed upon beforehand.

The procedure will be performed in Price B110 and the respective lab will be contacted to pick up the vasectomized males. We will not house vasectomized males in our animal room. After a two-week recovery period, the males can be used.

back to top

Rederivation

The Transgenic Facility provides rederivation services to all AECOM investigators seeking to maintain or save cell lines/strains. We advise that individual labs refrain from waiting until candidate mice are over 4 months, as advanced age decreases the chances of rederivation success. Investigators must contact the Transgenic Facility Director to schedule the procedure as well as discuss the details of candidate cell lines/strains and obtain and complete a Transgenic Rederivation Form. Scheduling is on a first come-first served basis.

Individual investigators are responsible for the super ovulation and mating of candidate mice. Once a target date has been agreed upon, investigators must obtain super ovulation hormones and a tube of embryo-specific media from the facility. Information on super ovulation scheduling is provided. Training for either plug checking or oviduct isolation can also be arranged with the facility director. The super ovulation hormones used are Pregnant Mare Serum (PMS) and Hydrochorionic Gonadotropin (HCG). PMS is distributed in the form of a frozen 50 IU microcentrifuge tube. HCG is distributed in the form of a 50 IU lyophilized powder in a microcentrifuge tube. Keep in mind when scheduling the injections that you must give the first injection (PMS) three days before the agreed upon target date for the rederivation procedure.

Immediately after HCG injection, set up your mating, preferably a single cage with one hormonally treated female with one male. No more than two females per male is recommended. The next morning check to see if any females are plugged and separate any unplugged females. If any super ovulated female is not plugged at this point, they will not be able to plug for at least 5-7 days, therefore it is advised that they be removed and set aside to recover if they need to be used again. Isolate the oviducts of all plugged females and place dissected oviducts into a Petri dish containing the embryo-specific media. Be very careful to not touch the dish with anything that touched the animals, change gloves frequently during the procedure. Wipe the dish with 10% bleach and a member of your lab that has had no contact with the animals or has been in any animal room must take the dish directly to Price B110. NO ONE WHO HAS HAD CONTACT WITH ANIMALS OR HAS BEEN IN ANY ANIMAL ROOM WITHIN THE PAST 24 HOURS IS NOT ALLOWED INTO THE MAIN FACILITY LABS IN Price B110. Facility personnel will then dissect the oviducts, isolate the embryos, and transfer them into a CD1 foster mother. The facility will then call your lab to pick up the cage(s) of foster mothers. The facility cannot house any cages after rederivation. After 19-20 days, foster mothers should give birth to pups. Please contact the facility and inform us of the number of pups born.

back to top

In-vitro Fertilization

Labs seeking to arrange In-vitro fertilizations with the facility must clarify what is needed in terms of animals, training and hormones weeks prior to the desired date of procedure and complete and submit an In-vitro Fertilization Form. All procedure dates must be agreed upon at the very least, 2 weeks ahead of target date. Individual labs must secure space to house animals either in their current respective animals rooms or arrange for housing with the Institute of Animal Studies in AECOM.

Individual investigators are responsible for the super ovulation of candidate mice. Once a target date has been agreed upon, investigators must obtain super ovulation hormones and a tube of embryo-specific media from the facility. Information on super ovulation scheduling is provided. Training for either plug checking or oviduct isolation can also be arranged with the facility director. The super ovulation hormones used are Pregnant Mare Serum (PMS) and Hydrochorionic Gonadotropin (HCG). PMS is distributed in the form of a frozen 50 IU microcentrifuge tube. HCG is distributed in the form of a 50 IU lyophilized powder in a microcentrifuge tube. Keep in mind when scheduling the injections that you must give the first injection (PMS) three days before the agreed upon target date for the In-vitro fertilization procedure.

The day of the procedure, investigators must bring either their dissected oviducts, males or both to Price, Rm B110. Be advised that it is preferable that two males per line are provided. NO ONE WHO HAS HAD CONTACT WITH ANIMALS OR HAS BEEN IN ANY ANIMAL ROOM WITHIN THE PAST 24 HOURS IS NOT ALLOWED INTO THE MAIN FACILITY LABS IN PRICE B110. Facility personnel will extract the sperm from the males, isolate the oocytes from the dissected oviducts, and then incubate the oocytes and sperm overnight in a CO2 incubator. The following day any fertilized eggs will be transferred into pseudopregnant CD1 females (20 maximum per female) and the respective lab will be called for cage pickup. Housing for this resulting cage is entirely the responsibility of the client lab, the facility will not house any cages under any circumstances. After 19-20 days, foster mothers should give birth to pups. Please contact the facility and inform us of the number of pups born.

back to top

Embryo Cryopreservation

Labs seeking to preserve mouse-lines must complete and submit an Embryo Cryopreservation Form. All procedure dates must be agreed upon at least two weeks prior to the desired procedure date. Individual labs must store the resulting cryogenic tubes of embryos in their own liquid-nitrogen tanks, the facility will not store any tubes.

Individual investigators are responsible for the super ovulation of candidate mice. Once a target date has been agreed upon, investigators must obtain super ovulation hormones and a tube of embryo-specific media from the facility. Information on super ovulation scheduling is provided. Training for either plug checking or oviduct isolation can also be arranged with the facility director. The super ovulation hormones used are Pregnant Mare Serum (PMS) and Hydrochorionic Gonadotropin (HCG). PMS is distributed in the form of a frozen 50 IU microcentrifuge tube. HCG is distributed in the form of a 50 IU lyophilized powder in a microcentrifuge tube. Keep in mind when scheduling the injections that you must give the first injection (PMS) six days before the agreed upon target date for the procedure. This is necessary because the facility performs 4-8 cell stage cryopreservation of embryos. Immediately following HCG injection, investigators must set up mating, a maximum of 2 females per male. The next morning isolate plugged mice, and 48 hours later isolate oviducts and bring them to Price Rm B110. NO ONE WHO HAS HAD CONTACT WITH ANIMALS OR HAS BEEN IN ANY ANIMAL ROOM WITHIN THE PAST 24 HOURS IS NOT ALLOWED INTO THE MAIN FACILITY LABS IN PRICE B110. The facility will isolate embryos, freeze 30 embryos per cryogenic vial, and then contact the respective lab for pickup.

There is no guarantee of success and labs must cryopreserve their own embryos.

back to top

Sperm Cryopreservation

Labs seeking to preserve mouse-lines must complete and submit an Sperm Cryopreservation Form. All procedure dates must be agreed upon at least two weeks prior to the desired procedure date. Individual labs must store the resulting cryogenic tubes of embryos in their own liquid-nitrogen tanks, the facility will not store any tubes.

The facility recommends two males per line, and that the males be within the range of 6-8 weeks of age. On the day of the procedure investigators must bring the males to Price Rm B110. NO ONE WHO HAS HAD CONTACT WITH ANIMALS OR HAS BEEN IN ANY ANIMAL ROOM WITHIN THE PAST 24 HOURS IS NOT ALLOWED INTO THE MAIN FACILITY LABS IN PRICE B110. The facility will dissect the males, isolate the sperm, freeze 15 cryogenic vials (the vials will contain the mixed sperm of the two males per line), and then contact the respective lab for pickup.

There is no guarantee of success and labs must cryopreserve their own vials of sperm.

back to top

Cancer
Center