Transgenic Services

• General Rules 
• Service Charges 
• Pronuclear Injection 
• Lentiviral Transgenesis 
• Rederivation 
• In-vitro Fertilization 
• Embryo Cryopreservation 
• Sperm Cryopreservation 
• Ovary Transplant 
• Miscellaneous Service 

General Rules

The Transgenic Facility services the entire research community of Albert Einstein College of Medicine and routinely provides transgenic mice to many labs. 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 located in Price B110 if they have been in contact with any animals within the past 24 hours.

No one under any circumstance is allowed to enter the facility animal room outside of facility personnel. No animals from any other animal room are allowed to be housed in the facility animal room. 

Lab personnel who are bringing animals for an agreed upon procedure must bring animals directly to Price B110. The lab member bringing the cage of animals must not have had any direct contact with the animals. If investigators are bringing animals to the Price Center from another building, they must obtain the proper cage transfer paperwork from Institute of Animal Studies in order to clear building security.

Pronuclear Injection

Applicants must contact the facility director and discuss their construct and expected phenotypes. Investigators must complete and submit an official Pronuclear Injection Form (which includes 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 100ug 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 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). Cut out the gel slice containing the DNA insert and submit to the facility. We will then purify the DNA.

Standard pronuclear injections use FVB embryos by default unless another strain is specifically requested. Injection scheduling is determined on a first come, first served basis unless extraordinary circumstances exist.

Nineteen to twenty days following injection, pups are born and kept in facility housing for an additional 10-14 days. They are then transferred to the investigator's animal room through the 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.

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.

Investigators must complete and submit an official Pronuclear Injection Form (which includes 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.

Investigators must specify the strain they want injected and discuss with the facility director and additional costs that are to be incurred.

Injections are scheduled on a first come, first served basis unless extraordinary circumstances exist.

Nineteen to twenty days following injection, pups are born and kept in the facility animal room for an additional 10-14 days. They are then transferred to the investigator's animal room via Institute of Animal Studies.

Rederivation

The Transgenic Facility provides rederivation services to all AECOM investigators seeking to maintain or save cell lines/strains. We advise individual labs to refrain from waiting until all candidate mice are over the age of 4 months, as advanced age decreases the chances of rederivation success. Investigators must fill out a Rederivation Form and contact the Transgenic Facility Director to schedule the procedure and discuss the details of candidate cell lines/strains. 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 can be found here. 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 hydrolyzed 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. 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. It is recommended you set up a maximum of two females for every male. The following morning, check to see if any females are plugged and separate any unplugged females from the males. If any super ovulated female has not plugged, 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. Isolate the oviducts of all plugged females and place dissected oviducts into a Petri dish containing embryo-specific media (M2, FHM). 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 while being careful to not use excessive bleach so as to compromise the embryos in the dish.

Assign a member of your lab, that has not had any contact with animals or been in an animal room, to 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 allowed into the facility labs in Price B110.

Facility personnel will then dissect the oviducts, isolate the embryos and transfer them into a CD1 foster mother. After the embryo transfer is complete the facility will call your lab to pick up the cage(s) of foster mothers. We cannot house any cages for investigators once the rederivation is complete. It is the responsibility of individual investigators to secure space in an animal room, via Institute of Animal Studies, before requesting a rederivation. In 19-20 days, foster mothers should give birth. Please contact the facility and inform us of the number of pups born.

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 cage(s) of animals resulting from the procedure, either within their current animal rooms or through arrangements with the Institute of Animal Studies.

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 is provided here. 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 hydrolyzed 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. Keep in mind when scheduling super ovulation 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 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 previous 24 hours is not allowed into the main facility labs in Price B110. Facility personnel will extract the sperm from the males, isolate 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 and the respective lab will be called to pick up their cage(s). After 19-20 days, foster mothers should give birth. The facility would appreciate it if client labs informed us of the number of pups born.

Cage housing is the responsibility of the client labs, the facility will not house any cages under any circumstances. Please contact the Institute of Animal Studies and obtain space in a room (if needed) prior to the date of the procedure.

Embryo Cryopreservation

Labs seeking to preserve mouse lines must complete and submit an Embryo Cryopreservation Form and discuss scheduling with the facility director. 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 can be found here. Training for plug checking or oviduct isolation can also be arranged with the facility director. The super ovulation hormones used are Pregnant Mare Serum (PMS) and hydrolyzed 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. Since the facility performs 4-8 cell stage cryopreservation, the first super ovulation hormone must be administered six days before the agreed upon date of the procedure.

Immediately after the second super ovulation injection (HCG), investigators must set up mating(s). A maximum of two females per male is recommended, with females being placed into the male's cage. The next morning, check for plugs and isolate the plugged mice. Two days later, isolate oviducts from the plugged mice and bring them to Price B110. No one who has been in 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 per cryogenic vial, and then contact the respective lab for pickup. There is no guarantee of success and labs must store their own cryogenic vials.

Sperm Cryopreservation

Labs seeking to preserve mouse-lines must complete and submit a Sperm Cryopreservation Form and discuss scheduling with the facility director. Individual labs must store their own resulting cryogenic tube, the facility will not store any tubes.

We recommend labs to provide two males per line and that males be within 4-5 months old. On the day of the procedure investigators must bring the males to Price B110. No one who has had contact with animals or has been in any animal room within 24 hours is allowed into the main facility labs in Price B110. The facility will dissect the males, isolate the sperm, freeze ~ 15 cryogenic vials of sperm (the vials will contain the mixed sperm of the two males per line), and the contact the respective lab for pickup. there is no guarantee of success and labs must cryopreserve their own vials of sperm.

Ovary Transplants

Labs seeking to save a line through an ovary transplant must contact the facility director to arrange scheduling and submit a completed Ovary Transplant Form.

On the day of the procedure, a member of the lab must bring the donor female to Price B110. No one who has had direct contact with any animals or been in any animal room within 24 hours is allowed into the main facility in Price B110. If the mice are housed in a building other than the Price Center, investigators must secure the necessary paperwork to document cage transfers through Institute of Animal Studies (IAS). Security personnel are instructed to stop any individual who is leaving the building with animals and require the individual to provide paperwork demonstrating IAS approval of the cage transfer.

The facility will excise the ovary(ies) of the donor female and transplant sections of the ovary into several recipient females. Once the procedure is complete, the respective lab will be contacted to pick up the cage(s) of mice. The facility does not store any cages of ovary transplant recipients under any circumstances. The requesting labs must set aside space in their personal animal rooms, or obtain space through Institute of Animal Studies before the date of the procedure.

Miscellaneous Service

If investigators seek reagents, assistance with mouse identification, dissection training, or any additional service the facility can provide that is not explicitly mentioned above, please feel free to contact the facility director to determine the feasibility of requests and submit a completed Miscellaneous Service Form.

Please remember that if anyone needs to come to the facility main labs in Price B110, that they should not have had any direct contact with animals or been in any animal room for 24 hours.