General Pronuclear Injection Questions
Q: What mouse strain does the facility use for pronuclear injection?
A: The facility uses FVB mice by default for pronuclear injections. If you need another strain then you will need to make special arrangements with the facility. Using an alternate strain may result in increased costs as the facility will need to purchase mice from a vendor and as a consequence will delay injection date as well. Any additional costs for housing and purchase of special mouse strains will be passed on to the individual lab making the request.
Q: How long does it take to schedule an injection?
A: Injection scheduling is based on a first come, first served basis. Historically it can be anything between a month to several months. Also bear in mind there are no injections scheduled on major holidays. Special requests made with respect to animal strains may delay date of injection. Please discuss scheduling issues with Dr. Ken Chen, the facility director.
Q: How long does the entire process take?
A: Once embryos are microinjected pups are born approximately 19-21 days after. Ten to 14 days after the birth of pups, candidate mice are transferred into the investigator's specified animal room.
Q: What are my responsibilities if I want to submit a construct for injection?
A: The person in the lab that will be the official contact person for the construct must contact Dr. Ken Chen to arrange scheduling. Download a copy of the Pronuclear Injection Form, complete and submit at Price B110. Obtain all the necessary clearances from Environmental Health and Safety for your construct and secure some space in your animal room or in one of the Institute for Animal Studies's quarantine rooms. Submit a 100µg slice of DNA from an Endofree-Qiagen preparation. The facility will then inject your construct and contact you when the animals are ready to be transferred to your animal room.
Q: Does your facility inject BAC DNA?
A: Yes we do. Please discuss with Dr. Ken Chen about the specific concerns with respect to using this type of DNA.
Q: If I am an AECOM investigator, how do I obtain my candidate mice?
A: Fourteen days following the birth of pups, the facility will fill out the appropriate paperwork with the Institute for Animal Studies and arrange for the transfer of cages to the investigator's specified animal room. The Transgenic Facility will call or email the contact person to inform them of the impending cage transfer. Please be advised that if the location of your animal changes for any reason it is the responsibility of the individual lab to inform the Transgenic Facility of the new animal room location.
Q: Does your facility house mice for investigators past weaning age?
A: No. Space in the facility animal room is very limited, we regret that we cannot house any cages for any investigator past 14-20 days post birth. It is the responsibility of the individual labs to secure space in an animal room with the Institute for Animal Studies (IAS) prior to submitting a construct. Please check the 'Contacts' link in the upper left menu for whom to contact with respect to securing animal room space.
Q: How many founders should I expect from an injection?
A: A pronuclear injection using the default facility strains will generally yield 10% founders. Facility policy is that we provide either 2 founders or 60 pups, which ever comes first.
Q: How do I know the age of my pups?
A: The date of birth of the pups in each cage is printed on the cage card.
General Superovulation Questions
Q: What is superovulation?
A: Superovulation is the controlled hyperstimulation of the ovaries to produce a greater number of oocytes. The two hormones used are Pregnant Mare Serum (PMS) and hydrolyzed Chorionic Gonadotropin (hCG). PMS is administered first, at 4PM, via intraperitoneal injection (IP). Forty-six hours later, hCG is given via intraperitoneal injection (IP).
Q: What hormones are used in superovulation?
A: The two hormones used are Pregnant Mare Serum (PMS) and hydrolyzed Chorionic Gonadotropin (hCG).
Q: Where can I obtain these hormones?
A: The facility can provide investigators with both hormones for a nominal fee. PMS is given in 1ml aliquots of 50IU each, already diluted with DDH2O. Each mouse receives 5IU so give each mouse 0.1cc of the solution. hCG is given in lyophilized aliquots of 50IU each. Dissolve the hCG in 1ml DDH2O or saline when you are ready to inject. Store both hormones in -70 degrees Celsius. The PMS needs to be used within 2 weeks of dilution. The hCG can be stored indefinitely.
Q: What is the superovulation scheduling for a rederivation?
A: Single cell embryos are needed for rederivations, so three days before the agreed date of the procedure, PMS at 4PM and hCG 46 hours later (2PM). Immediately after hCG injection set up mating. For your mating, place 1-2 superovulated females into a cage housing a single male. The next morning, check for plugs and dissect out the oviducts of the plugged out. Bring the oviducts to the facility.
Q: What is the superovulation scheduling for an embryo cryopreservation?
A: Embryos in the 4-8 cell stage are required for embryo cryopreservations. Four days prior to the agreed date of the procedure, PMS at 4PM and hCG 46 hours later (2PM). Immediately after hCG injection set up mating. For your mating, place 1-2 superovulated females into a cage housing a single male. The next morning, check for plugs and separate plugged from unplugged females. The day after plug checks, bring the plugged females to the facility.
- top-
General Rederivation Questions
Q: How long does it take to schedule a rederivation?
A: Rederivations, like all other services, are scheduled on a first come-first served basis. Please discuss all scheduling with Dr. Ken Chen.
Q: How long does the entire process take?
A: Embryo collection and oviduct transfer takes a single day. Approximately 20 days after, pups are born. Birthrate is highly dependent on the condition of the embryos when transferred. It is highly recommended that labs superovulate females to increase embryo yield. Hormones are provided by the facility. Investigators must superovulate the desired mice to be rederived, facility personnel are not allowed to go into another animal room to help with this. For questions regarding superovulation please see the FAQ section on superovulation.
Q: What is the facility policy regarding potentially contaminated mice?
A: Because of the large number of animals the facility distributes to the college, we must exercise extreme caution and assume all animals that come from other labs to be potentially contaminated. Out of safety for all the investigators we service we cannot make any exceptions. In light of this, investigators requesting rederivations must perform some preliminary dissection at their lab and then bring the dish containing the dissected oviducts to the facility labs. The lab member who brings the dish containing the dissected oviducts must not have had any contact with any animals or have been in any animal room for the previous 24 hours.
Q: How will the delivery of mice to be rederived be handled?
A: Anyone who has had contact with animals or been in any animal room is not allowed into the facility labs in Price B110. On the day of the rederivation, a second lab member who satisfies the aforementioned requirement must bring the dissected oviducts to Price B110. Before the dish containing oviducts is to be transported to the facility labs, make sure you wipe the dish with paper towel moistened with bleach. Do not use excessive amounts of bleach and risk introducing bleach to the media inside the dish.
Q: Do I need to superovulate mice for this procedure?
A: It is highly recommended you superovulate your females in order to increase embryo yield and your chances of a successful rederivation. Hormones are provided with the procedure, please schedule a pickup time with the facility. Recommended pickup time should be 5-7 days before administering the first dose (PMS). Due to the relatively short life of the PMS solution the facility keeps on hand, a small stock is kept, so please do not wait the day before your first dosing to arrange for hormone pickup. For questions regarding superovulation please see the Superovulation FAQ on this page.
Q: Where can I get training for the surgical techniques I am expected to perform?
A: Labs requesting rederivation must be able to do dissect out the oviducts from mice. Please schedule oviduct dissection training with the facility director well in advance of the date of the procedure.
Q: Where do I bring the oviducts on the day of the procedure?
A: Bring dissected oviducts directly to Price B110. The lab member who brings the dish must have had no contact with animals or have been in any animal room for the past 24 hours. Make sure that before you come to the facility, the dish has been wiped with paper towel moistened with 10% bleach. Careful not to use an excessive amount of bleach and risk it seeping into the contents of the dish.
Q: Can the facility house foster mothers from the rederivation?
A: No. After the procedure the respective lab will be contacted to pick up the cage containing the foster mothers. It is the responsibility of requesting labs to secure space in their own animal room or in a quarantine room provided by Institute for Animal Studies (IAS). Please check the 'Contacts' link in the upper left menu for whom to contact with respect to securing animal room space.
Q: When will pups be born if the rederivation is successful?
A: Pups should be born approximately 20 days after the oviduct transfer. Rederivation success is highly dependent on the condition of the embryos transferred into the pseudo pregnant females.
- top -
General In Vitro Fertilization Questions
Q: How long does it take to schedule an in vitro fertilization?
A: In vitro fertilizations, like all services are scheduled on a first come, first served basis. Please contact the facility director arrange scheduling.
Q: How long does the entire process take?
A: The isolation of oocytes and sperm, incubation, and the transfer of divided embryos into recipient females takes two days.
Q: What is the facility policy regarding potentially contaminated mice?
A: Due to the large number of mice the facility distributes throughout the college, we must exercise extreme caution and assume all mice from other labs to be potentially contaminated, no exceptions. In light of this, investigators requesting IVFs must perform some preliminary dissection at their lab and then bring the dish containing the dissected tissue to the facility labs. The lab member who brings the dish must not have had any contact with animals or been in an animal room for the previous 24 hours.
Q: Do I need to superovulate mice for this procedure?
A: If you lab is providing the oocytes then yes, you should superovulate your females. Hormones are provided by the facility, please pickup a set well in advance of the procedure date. For questions regarding superovulation, check the section in this FAQ titled 'General Superovulation Questions'.
Q: Can the facility provide donor males or females?
A: The facility can provide donor males or females at cost.
Q: What are my responsibilities for an IVF?
A: Labs must provide either the sperm or the oocytes for the procedure. If the sperm or the oocytes is not frozen, then training for cauda epididymis + vas deferens dissection or oviduct dissection may be needed. Please schedule the appropriate training with the facility director well in advance of the procedure date. On the day of the procedure, bring the relevant dissected tissue to Price B110. Facility policy is that anyone who has had contact with animals or been in an animal room for the past 24 hours is not allowed into the facility labs. Labs must coordinate with their personnel to have an individual, who does not violate this rule, deliver the dish containing dissected tissue. Please remember to wipe the dish with paper towel moistened with 10% bleach before you come to Price B110. Be careful not to use excessive bleach and risk bleach seeping into the contents of the dish. If the sperm or oocytes being provided by the lab is in a frozen vial(s), then please submit the vials well in advance of the scheduled procedure date.
Q: What is the facility success rate for in vitro fertilization?
A: Using FVB oocytes and sperm from mice 6-8 weeks of age, the facility success rate is 90%. Different strains and donor mice older than 6-8 weeks of age have varying effects on IVF success.
Q: Can the facility house the foster mothers resulting from the procedure?
A: Generally no, after the procedure the respective lab will be contacted to pick up the cage containing the recipient females. An exception is made if what is provided to the facility are vials of frozen sperm. The cage(s) could then be housed in the facility animal room for monitoring until 10-14 days past birth of pups. It is the responsibility of requesting labs to secure space in their own animal room or in a quarantine room provided by Institute for Animal Studies (IAS). Please check the 'Contacts' link in the upper left menu for whom to contact with respect to securing animal room space.
Q: When will pups be born if the IVF is successful?
A: Recipient females should give birth approximately 20 days following oviduct transfer into CD1 pseudo-pregnant females. IVF success is dependent on the health of the sperm or oocytes given for the procedure.
- top -
General Cryopreservation Questions
Q: What is expected of me once a procedure date has been agreed to?
A: On the day of the procedure, labs must bring labelled cryogenic vials for each line, 10 vials per line. Additional investigator responsibilities are dependent on whether embryos or sperm is being frozen. For sperm freezings, investigators must be familiar with cauda epididymis + vas deferens dissection and if not, schedule for training well in advance of the procedure date. For embryo freezings, investigators must be familiar with plug checking as well as oviduct dissection and if not, schedule for training well in advance of procedure date. Embryo freezings also require superovulation and investigators must also arrange for hormone pickup at least a week before scheduled procedure date. On the day of the procedure, the lab member assigned to bring the dish containing either the cauda epididymis + vas deferens or the oviducts must not have had any contact with animals or been in an animal room for the past 24 hours. Be sure to wipe the dish with paper towel moistened with 10% bleach before bringing the dish to Price B110. Do not use an excessive amount of bleach so as to risk it seeping into the contents of the dish.
Q: Do you freeze embryos as well as sperm?
A: Yes, we can freeze either sperm or embryos, but only one or the other per procedure date. The facility freezes embryos at the 4-8 cell stage.
Q: How many males should I bring for a sperm freezing?
A: Two males per line is preferred, one line per procedure date.
Q: How should I set up matings if I need to bring fertilized eggs?
A: Once the second hormone (hCG) is given to your females, you can immediately set up matings. Ideally, place 1-2 females into a cage housing a single male. The next morning, check for plugs and separate plugged females from unplugged females. Since the facility freezes embryos at the 4-8 cell stage, bring the plugged mice to the facility 2 days following the discovery of the plug(s). So to recap, if your procedure is an embryo cryopreservation and it is scheduled on, for example, January 10th, then you must PMS your females on January 5th, hCG January 7th and set up matings immediately after hCG, check plugs January 8th and end mating, and finally dissect out the oviducts of the plugged mice on January 10th and bring to the facility.
Q: How long does it take to schedule a cryopreservation?
A: Cryopreservations, like all services, are scheduled on a first come, first served basis. Please contact the facility director to discuss scheduling.
Q: How long does the entire procedure take?
A: The procedure can take a full day. Embryo cryopreservation also requires the use of a controlled rate freezer to incrementally lower the temperature of the embryos to -80° C, a process that takes several hours. Labs must pickup their vials the following mornings at Price B110. Remember anyone who has been in contact with animals or been in an animal room within 24 hours is not allowed to enter the facility main labs at Price B110.
Q: What is the facility policy regarding contaminated mice?
A: Because the facility distributes a large number of animals throughout the college, me must exercise extreme caution and assume all animals from other labs to be potentially contaminated, no exceptions. In light of this, investigators requesting sperm or embryo cryopreservation must perform some preliminary dissection at their labs and then bring the dissected tissue to the facility. The lab member bringing the dish must have not had contact with any animals or been in an animal room for the previous 24 hours.
Q: Do I need to superovulate mice for this procedure?
A: If the lab is requesting an embryo cryopreservation then yes, you need to superovulate mice. Refer to the section in this FAQ titled 'General Superovulation Questions' for specifics on hormone scheduling for your procedure. We regret we cannot help investigators with this aspect of the service, a precaution taken in order to safeguard the integrity of the facility animal room and the personnel who access it regularly.
Q: How many females should I superovulate for an embryo freezing?
A: To be safe, superovulate 10-20 females per line for each embryo freezing, one line per procedure. Refer to the section in this FAQ titled 'General Superovulation Questions' for specifics on hormone scheduling for your procedure. Hormones are provided with the procedure, please schedule hormone pickup well before the scheduled date of the first dosing (PMS).
Q: What is the facility success rate for sperm or embryo cryopreservation?
A: If the donor males are 4-5 months old, relatively healthy, or the sperm vials were properly maintained in storage using liquid nitrogen, then the success rate is nearly 95%. If the frozen embryos were from a relatively healthy donor females 4-6 weeks of age, or the embryo vials were properly maintained in storage using liquid nitrogen, then the success rate is nearly 90%.
Q: Can the facility store the resulting cryogenic vials for my lab?
A: No, it is the responsibility of the individual labs to store their own cryogenic vials. It is advised you store the vials in liquid nitrogen. After completion of the procedure your lab will be contacted to pickup the resulting vials.
- top -
General Ovary Transplant Questions
Q: What is the purpose of an ovary transplant?
A: The purpose of an ovary transplant is to preserve the reproductive viability of a mouse line. Sometimes, a lab may find an important mouse line reduced to a sickly or dying female. An option to save the line would be to dissect out the ovaries of the donor mouse and implant them into recipient females of the same background. The recipient female's own ovaries would be removed initially and the donor's would be implanted. After a period of recovery, the recipient female has a good chance to produce eggs genetically consistent with the donor female.
Q: What is the facility policy regarding potentially contaminated female candidates?
A: Because the facility distributes a large number of animals throughout the college, we must exercise extreme caution and assume all animals from other labs to be potentially contaminated, no exceptions. In light of this policy, investigators requesting ovary transplants must perform ovary dissections at their own labs and bring the dissected ovaries to the facility labs. The individual bringing the dish to the facility labs must additionally have not had any contact with animals or been in an animal room for the previous 24 hours.
Q: What are my responsibilities for this procedure?
A: Once a procedure date has been agreed to, investigators should schedule ovary dissection training with the facility director (if needed). Discuss with the facility director the strain of recipient females you will need. It is possible the facility may not have the desired strain on hand and will need to order it from a vendor, something that may delay the date of the procedure. On the day of the procedure, a lab member should dissect out the ovaries, one line per dish. Have a second lab member, one who has not had any contact with animals or been in any animal room the previous 24 hours, bring the dish(es) containing the ovaries to the facility labs in Price B110. Please make sure that before you come to the facility, wipe the dish in paper towel moistened with 10% bleach. Be careful not to use excessive amounts of bleach in order to avoid any seeping into the contents of the dish. Once the facility performs the procedure it will contact the lab and request a lab member to pickup the cage(s) containing the recipient females.
Q: How long does it take to schedule an ovary transplant?
A: Ovary transplants are typically scheduled on a first come, first served basis, but exceptions can be made if there is an emergency situation. Please discuss feasibility with the facility director.
Q: How long does the procedure take?
A: The procedure should take no longer than an hour or two, depending on the number of female donors submitted. If investigators deliver the ovaries early enough in the day, you should be able to collect the cages of recipient females at the end of the day. The facility will contact you once the procedure is complete for cage pick up.
Q: How many donor females must I provide for the procedure?
A: One to three donor females is recommended for each line.
Q: Can the facility provide the recipient females?
A: Yes, recipient females can be purchased by the facility at cost. Please discuss with the facility director the requisite background for your line.
Q: Can the facility house the recipient females for my lab?
A: No. After the procedure the respective lab will be contacted to pick up the cage(s) containing the recipient females. It is the responsibility of requesting labs to secure space in their own animal room or in a quarantine room provided by Institute for Animal Studies (IAS). Please check the 'Contacts' link in the upper left menu for whom to contact with respect to securing animal room space.
- top -
General Questions about Rescuing a Mouse Line
Q: My mouse is not mating, what can you do to help?
A: If you have set up matings, checked for plugs every morning, and have failed to find a plug, it is possible there is some physical reason the mouse cannot mate or the mouse is sterile. Consider attempting an IVF. Discuss the specifics of your problem with the facility director. For additional questions about IVFs, check the section in this FAQ titled 'General IVF Questions'.
Q: My mouse line X is infected with pathogen Z, what are my options?
If your mice is infected with a pathogen then a rederivation is recommended if you have both males and females of line X to mate and generate the embryos needed to transfer into a recipient female. If you have only a male(s) or a female(s), then an IVF is your only option. Discuss the specifics of your problem with the facility director. For additional information on rederivations, check the section of this FAQ titled 'General Rederivation Questions'. For additional information on IVFs, check the section of this FAQ titled 'General IVF Questions'.
Q: I need to generate mice of line X and I have a few vials of sperm/embryos, can you help?
A: The facility can help with this. You can bring us the vials of whatever you have available, be it sperm or embryos, and we will either perform an IVF (sperm) or an oviduct transfer (embryos). Keep in mind that if you provide sperm, there will be an additional charge for the facility to provide the eggs needed in the IVF. Discuss with the facility director the specifics of your problem. For additional information on IVFs, check the section in this FAQ titled 'General IVF Questions'. Transferring frozen embryos into a recipient female is treated as a Miscellaneous Service.
Q: How fast can you help me rescue my line? I am afraid the mouse will die soon.
A: Contact the facility director and discuss the specifics of your situation. Emergency situations are given special consideration where scheduling is concerned.
Q: Can you help me check if my mice are plugging?
A: Because the facility distributes a large number of mice throughout the college, we must exercise extreme caution and assume all animal from other labs to be potentially contaminated, no exception. We regret we cannot help by physically going to your animal to check plugs. We are happy to provide training on how to check plugs or examples of plugs. If you want training please contact the facility director to schedule a session.
Q: If the facility can attempt a procedure to rescue a line, can the facility house the resulting cages of mice in their animal room?
A: No. It is the responsibility of the individual labs to secure space in their own animal room or in a quarantine room provided by Institute for Animal Studies (IAS). Please check the 'Contacts' link in the upper left menu for whom to contact with respect to securing animal room space.
- top -
General Miscellaneous Service Questions
Q: What kinds of miscellaneous services does your facility provide?
A: We can provide
- superovulation hormones
- mouse vasectomies
- transfer of embryos into recipient females
- mouse IV (tail) injections
- mouse egg/sperm/embryo isolation
Please contact the facility director for further details.
Q: What kinds of training does your facility provide?
A: We can provide
- pronuclear injection training
- lentiviral transgenesis training
- mouse vasectomy training
- mouse egg/sperm/embryo isolation training
- mouse IV injection training
- plug checking training
- oviduct transfer training
Please contact the facility director to schedule a training session. Please be aware that on the day of the training session, facility policy mandates all visitors to not have had any contact with animals or been in an animal room the previous 24 hours.
Q: Does your facility provide superovulation hormones?
A: Yes we do. We keep a small stock of PMS and hCG on hand. PMS is dissolved in DDH20, 50IU per ml. HCG is stored as a lyophilized powder, 50IU per tube. Please contact the facility director to schedule a pickup. For specifics on superovulation scheduling, please refer to the section in this FAQ titled 'General Superovulation Questions'.
Q: Who should I contact if I want to schedule a training session?
A: Contact the facility director to arrange scheduling. Please bear in mind some training requires the sacrifice of animals and as a result may incur a charge. Mice will be charged at cost.
Q: Can you help me with mouse egg/sperm/embryo isolation?
A: Yes. The facility distributes a large number of animals throughout the college and must exercise extreme caution and assume all animals from other labs as potentially contaminated. In light of this, we must ask investigators to perform some preliminary dissection before the facility can complete the procedure. In the case of egg or embryo isolation, investigators must dissect out the oviducts from their mice and bring to the facility a dish containing the oviducts. In the case of sperm isolation, investigators must dissect out the cauda epididymis and vas deferens of the males and bring the aforementioned to facility in a dish. In either case, the lab member who brings the dish to the facility labs must not have had any contact with animals or been in an animal room for the previous 24 hours. Be sure to wipe the dish with paper towel moistened with 10% bleach before bringing the dish to Price B110. Do not use an excessive amount of bleach so as to risk introducing it into the contents of the dish.
- top -
|
