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 it
may also 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
and during the week of the Annual
Mouse Developmental Genetics Course.
Also if any special request is made with respect to animal strain etc.
then the actual date of injection could be further postponed. Please
discuss with Dr.
Ken Chen, facility director for all scheduling matters.
Q: How long does the entire process take?
A: From the initial day of injection, approximately 35-40
days. 19 to 21 days following transfer into foster mother, pups are
born. The date of the transfer is written on the cage cards.
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 form
and have it filled out. 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
of Animal Studies quarantine rooms. Submit a 100µg slice of
DNA from an Endofree-Qiagen preparation. The facility will then inject
and contact you when the animals are to be shipped 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: When a Pronuclear
Injection Form is first submitted, the lab's animal room is specified
as an item in the form. Fourteen days following the birth of pups, the
facility will fill out the appropriate paperwork with Institute
of Animal Studies and have them transfer the cages to the investigator's
room. The Transgenic Facility will call you and inform the respective
lab that their mice is being transferred into their room. Please be
advised that if the location of your animal changes for any reason that
it is the responsibility of the individual lab to inform the Transgenic
Facility of the new location of your animal room.
Q: Does your facility house mice for investigators past weaning
age?
A: No. With the addition of new foster mothers with each injected
construct and the relative size of the facility animal room we cannot
house any cages for any investigator past 14-20 days. It is the responsibility
of the individual labs to secure space in an animal room with the Institute
of Animal Studies.
Q: How many founders should I expect from an injection?
A: A pronuclear injection using the default facility strains
and foster mothers 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: For the sake of facility record-keeping, the date of the
oviduct transfer is recorded on the label of the cage. Approximately
20 days after the transfer date is the expected date of birth.
General Embryonic Stem Cell
Injection Questions
Q: What mouse strain does the facility use for ES Cell injections?
A: By default ES cell injections use C57/B6 blastocysts and
CD1 foster mothers. If you wish to use any other strain please speak
to Mr.
Harry Hou Jr. to make arrangements.
Q: How long does it take to schedule an injection?
A: Injection is 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 and during the week
of the Annual
Mouse Developmental Genetics Course. Please discuss with Harry
Hou Jr. for all ES cell injection scheduling matters.
Q: How many days can I reserve for injection?
A: The facility generally reserves one week blocks for an individual
lab and within that week, there are a maximum of four days of injection.
Labs can choose to inject the same cell line for all four days, two
cell lines for two days each, or four cell lines one day each. It is
recommended that you inject each cell line at least two days to enhance
the possibility of obtaining chimeras.
Q: Where can I find information about how to prepare ES cells
for the injection?
A: Contact Harry
Hou Jr. and he can direct you to experienced researchers who have
successfully grew and maintained cell lines.
Q: If I am an AECOM investigator, how do I obtain my chimeras?
A: Approximately seventeen days after uterine transfer (immediately
following ES cell microinjection there must be uterine transfer) pups
should be born. The facility will then house the pups for 2-3 weeks
after birth and then submit the appropriate paperwork with Institute
of Animal Studies for a cage transfer. The destination room is the
same room specified by the respective investigator on the injection
form. Please be advised that it is the responsibility of the individual
lab to notify the facility if the lab animal room has changed.
Q: Does your facility house mice for investigators past weaning
age?
A: Because of the size of the facility animal room and the
demand for injections, we cannot keep an investigator's cages past 2-3
weeks after birth. It is the responsibility of individual investigators
to secure space in an animal room with Institute
of Animal Studies.
Q: How many chimeras can I expect from an injection?
A: Healthy and well maintained ES cells increase the likelihood,
but does not guarantee, the birth of chimeras. Chimera numbers are unpredictable
and can range from 2 chimeras from 4 days injection to 90% of all births.
Q: How do I know the age of my pups?
A: For facility record-keeping purposes, the date of the uterine
transfer is recorded on the cage card. Approximately 17 days after the
uterine transfer, pups are born.
Rederivation Questions
Q: How long does it take to schedule a rederivation?
A: Because of pronuclear injection scheduling, rederivations
are often scheduled on either Mondays, Wednesdays or Fridays. As with
the other services, they are offered on a first come first served basis.
Please discuss all scheduling with Dr.
Ken Chen.
Q: How long does the entire procedure take?
A: The harvesting of embryos, and the oviduct transfer takes
a single day. Approximately 20 days after transfer, pups are given birth.
Birthrate is highly dependent on the condition of the embryos when transferred.
It is recommended that labs super ovulate their females to increase
embryo yield. Hormones are provided by the facility for the procedure.
It is recommended that investigators schedule the administration of
the hormones (PMS and HCG) based on the expected date of the procedure
as instructed in the Super Ovulation Scheduling section of
the Services
and Preparation page.
Q: What is the facility policy regarding potentially contaminated
mice?
A: Because of the large amount of animals the facility distributes
to the college we must exercise extreme caution and assume all animals
that have not directly come from the facility animal room as potentially
contaminated...no exceptions. As a rule we require that the lab member
who has been in the respective lab's animal room do not come to the
facility main labs. A second lab member must bring dissected oviducts
directly to Price Rm. B110. Great care must be taken to avoid bringing
materials that have had contact with any animals. If contact is suspected,
carefully wipe the surface with 10% bleach. Oviduct isolation training
is available and if necessary, should be arranged upon agreement of
procedure date.
Q: Do I need to super ovulate mice to do this procedure?
A: It is highly recommended that you super ovulate your females
in order to increase embryo yield and your chances for 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 the date of administering the first dose (PMS). Due to the
nature of the relatively short life of the hormones and the controlled
stock do not wait until the day before the first dose to arrange for
hormone pickup. For questions about hormone dosage scheduling please
consult the Super Ovulation Scheduling section of the Services
and Preparation page.
Q: Where do I bring the rederivation candidates the day of
the procedure?
A: Bring dissected oviducts directly to Price Rm. B110. The
lab member who brings the dish must have had no direct contact with
the animals or been in any animal room the same day. The dish will contain
the dissected oviducts of the candidate mice in a special media that
will be provided by the facility. Please wipe the dish with 10% bleach
and then change your gloves before bringing the dish to the lab. Schedule
oviduct dissection training with the facility if needed.
Q: Can the facility house the foster mothers resulting from
the rederivation?
A: No. After the procedure the individual lab will be contacted
to pick up the cage containing the foster mothers.
Q: When will pups be born if the pregnancy is successful?
A: Approximately 20 days after the oviduct transfer pups should
be born. Rederivation success is highly dependent on the condition of
the embryos transferred into the pseudo pregnant females.
In vitro Fertilization Questions
Q: How long does it take to schedule an in vitro fertilization?
A: In vitro fertilizations are scheduled on either Mondays,
Wednesdays or Fridays, on a first come first served basis. Please discuss
all IVF scheduling with Dr.
Ken Chen.
Q: How long does the entire procedure take?
A: The isolation of oocytes and sperm, incubation of sperms
and oocytes, and the transfer of divided embryos takes two days. If
labs are providing the oocytes then it is advised that females are super
ovulated according to Super Ovulation Scheduling section of
the Services
and Preparation page. Hormones are provided by the facility as part
of the service. Due to the nature of the relatively short life of the
hormones and the controlled stock do not wait until the day before the
first dose to arrange for hormone pickup. If the respective lab provides
the sperm then the facility will super ovulate females from the facility
animal room.
Q: What is the facility policy regarding potentially contaminated
mice?
A: Because of the large amount of animals the facility distributes
to the college we must exercise extreme caution and assume all animals
that have not directly come from the facility animal room as potentially
contaminated...no exceptions. As a rule we require that the lab member
who has been in the respective lab's animal room do not come to the
facility main labs. A second lab member must bring dissected oviducts
directly to Price Rm. B110. If the respective lab is not providing
the oocytes but the sperm instead for the IVF, then just prior to the
time of the procedure, sacrifice the male and flush with 70% ethanol.
Then a second lab member immediately bring the sacrificed animals to
Price Rm. B110. Great care must be taken to avoid bringing materials
that have had contact with any animals. If contact is suspected, carefully
wipe the surface with 10% bleach. Oviduct isolation training is available
and if necessary, should be arranged upon agreement of procedure date.
Q: Do I need to super ovulate mice for this procedure?
A: If your lab is providing the oocytes then it is highly recommended
that you super ovulate your females according to Super Ovulation
Scheduling section of the Services
and Preparation page. Hormones are provided by the facility as part
of the service. Due to the nature of the relatively short life of the
hormones and the controlled stock do not wait until the day before the
first dose to arrange for hormone pickup. If the respective lab provides
the sperm then the facility will super ovulate females from the facility
animal room. Please note that any animals provided by the facility,
male or female, will be an additional charge and will be provided at
cost.
Q: Can the facility provide donor females?
A: The facility will provide donor females or males at cost.
Q: What is the facility success rate for in vitro fertilizations?
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 have varying affects on IVF success.
Q: Can the facility house the foster mothers resulting from
the procedure?
A: No. After the procedure the individual lab will be contacted
to pick up the cage containing the foster mothers.
Q: When will the pups be born if the pregnancy is successful?
A: Pups should be born approximately 20 days after the oviduct
transfer into pseudo-pregnant CD1 females. IVF success is dependent
on the health of the embryos and sperm used for the procedure.
Cryopreservation Questions
Q: Do you freeze embryos as well as sperm?
A: Yes, we freeze either sperm or embryos, but only one or
the other per procedure date. The facility freezes embryos at the 4-8
cell stage in order to increase the chance of cell line survival.
Q: How many males should I bring for a sperm freezing?
A: Two males per cell line is more than enough. One cell line
per procedure date preferred.
Q: How many females should I super ovulate for an embryo freezing?
A: To be safe, super ovulate 10-20 females per cell line for
each embryo freezing. One cell line per procedure is preferred. Schedule
your hormone dosing according to the Super Ovulation Scheduling
section of the Services
and Preparation page. Hormones are provided with the procedure.
Q: How should I set up matings if I need to bring fertilized
eggs?
A: Ideally you should house your males separately in his own
cage. Three days before the scheduled cryopreservation and right after
administering the HCG (second dose of super ovulation), add a maximum
of two super ovulated females per male. Never house more than one male
per cage during matings. Preferably you should add the females to the
cage with males and not add a male to a cage containing females. Also
it is very likely that once you separate your males into their own cages
you cannot recombine them into a single cage again. The next morning
after pairings check the females for a coital plug and separate the
plugged females into a cage. Remove all the females from the cages containing
males whether they are plugged or not. If a super ovulated female is
not plugged the day following pairings they will not be able to plug
for at least 10-14 days. If necessary, training to identify a coital
plug can be arranged with the facility.
Q: How long does it take to schedule a cryopreservation?
A: Cryopreservations are scheduled on either Mondays, Wednesdays
or Fridays on a first come first served basis. Please contact Dr.
Ken Chen for all cryopreservation scheduling.
Q: How long does the entire procedure take?
A: The procedure can take up to two days. The first say the
embryos or sperm is isolated and placed into a cooling device which
slowly lowers the temperature at a controlled rate. The following morning
(or if the procedure was started early enough, the evening of the same
day) the respective lab will be called up to pick up their vials of
frozen embryos or sperm.
Q: What is the facility policy regarding potentially contaminated
mice?
A: Because of the large amount of animals the facility distributes
to the college we must exercise extreme caution and assume all animals
that have not directly come from the facility animal room as potentially
contaminated...no exceptions. As a rule we require that the lab member
who has been in the respective lab's animal room do not come to the
facility main labs. A second lab member must bring dissected oviducts
directly to Price Rm. B110. If the respective lab is not providing
embryos but sperm instead, then just prior to the time of the procedure,
sacrifice the male and flush with 70% ethanol. Then a second lab member
immediately bring the sacrificed animals to Price Rm. B110. Great care
must be taken to avoid bringing materials that have had contact with
any animals. If contact is suspected, carefully wipe the surface with
10% bleach. Oviduct isolation training is available and if necessary,
should be arranged upon agreement of procedure date.
Q: Do I need to super ovulate mice for this procedure?
A: If you are providing 4-8 cell stage embryos then yes, it
is recommended you super ovulate your females. Administer your dosages
according to the Super Ovulation Scheduling section of the
Services
and Preparation page. Hormones are provided with the procedure.
Due to the nature of the relatively short life of the hormones and the
controlled stock do not wait until the day before the first dose to
arrange for hormone pickup.
Q: What is the facility success rate for sperm or embryo cryopreservation?
A: If the donor males are 6-8 weeks of age, relatively healthy,
and the sperm vials were properly maintained in storage using liquid
nitrogen then the success rate is nearly 95%. If the frozen embryos
were from relatively healthy donor females 4-6 weeks of age, and the
embryo vials were 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. Preferably store the vials in liquid nitrogen.
After completion of the procedure your lab will be contacted to pickup
the resulting vials.
Miscellaneous Service Questions
Q: What must I do to obtain super ovulation hormones?
A: Rederivations, IVFs, and embryo cryopreservations provide
the requesting labs with the necessary doses to super ovulate the animals
to be used for the respective procedure. Simple schedule a pickup time
with the facility 5-7 days the date of the expected first dose. For
help in super ovulation scheduling consult the Super Ovulation Scheduling
section of the Services
and Preparation page. Two sets of hormones are administered, PMS
and HCG, each tube given will contain enough hormone for 10 animals.
Hormones are distributed only in such 10-dose tubes, we cannot distribute
them in requested ml volumes.
If you are not requesting the aforementioned services and need the
hormones for personal research purposes then you must fill out a Transgenic
Miscellaneous Service Form and arrange a pickup time. Be advised
there is a charge for the hormones.
Q: How long does it take to schedule a vasectomy?
A: Vasectomies are often scheduled on Friday afternoons on
a first come first served basis and should not take longer than one
day to complete. Please contact Dr.
Ken Chen to schedule a vasectomies. On the agreed upon procedure
date, bring the candidate males to Price Rm. B110 and leave the cage
in the room. Please see the next question in this FAQ regarding handling
of vasectomy candidates.
Q: What is the facility policy regarding potentially contaminated
male candidates?
A: Because of the large amount of animals the facility distributes
to the college we must exercise extreme caution and assume all animals
that have not directly come from the facility animal room as potentially
contaminated...no exceptions. As a rule we require that the lab member
who has been in the respective lab's animal room do not come to the
facility main labs. A second lab member must bleach the cage containing
the males, and bring the cage directly to Price Rm. B110.
Q: How do I arrange for some mouse identification?
A: Contact Dr.
Ken Chen to schedule mouse identification. You will need to fill
out a Transgenic
Miscellaneous Service Form and be advised there will be a service
charge.
Q: What methods of mouse identification do you provide?
A: The facility can provide toe clipping or ear notching as
methods for identification. The facility also offers tail clipping for
DNA processing purposes in conjunction with a form of identification.
Q: What must I do if I need to isolate some mouse embryos?
A: You can choose to either receive training for embryo isolation
or have the facility perform the procedure for you. Fill out a Transgenic
Miscellaneous Service Form and schedule either training or the service
with Dr.
Ken Chen. Be advised that the training is free, but there is a charge
if the facility performs the isolation.
If the facility will perform the isolation for you then please review
the facility policy with respect to animal handling and facility grounds.
Because of the large amount of animals the facility distributes to the
college we must exercise extreme caution and assume all animals that
have not directly come from the facility animal room as potentially
contaminated...no exceptions. As a rule we require that the lab member
who has been in the respective lab's animal room do not come to the
facility main labs. A second lab member must bleach the cage containing
the mice, and bring the cage directly to Price Rm. B110. If possible
we request that respective labs could do the oviduct isolation beforehand
and bring a dish containing only the dissected coils. Oviduct dissection
training is available free of charge and must be scheduled with Dr.
Ken Chen.
The whole procedure should not take longer than 3 hours (for 10 - 20
mice) and the respective lab will be contacted to pick up the dish of
isolated embryos.
Q: What must I do if I need to isolate some mouse blastocysts?
A: You can choose to either receive training for blastocyst
isolation or have the facility perform the procedure for you. Schedule
either training or the service with Harry
Hou Jr. Be advised that the training is free but there is a charge
if the facility performs the isolation.
If the facility will perform the isolation for you then please review
the facility policy with respect to animal handling and facility grounds.
Because of the large amount of animals the facility distributes to the
college we must exercise extreme caution and assume all animals that
have not directly come from the facility animal room as potentially
contaminated...no exceptions. As a rule we require that the lab member
who has been in the respective lab's animal room do not come to the
facility main labs. A second lab member must bleach the cage containing
the mice, and bring the cage directly to Price Rm. B110.
The whole procedure should not take longer than 3 hours (for 10 - 20
mice) and the respective lab will be contacted to pick up the dish of
isolated blastocysts.
Q: How do I schedule for assistance doing mouse intravenous
injections?
A: Contact Dr.
Ken Chen for IV injection training or for the facility to perform
the service. Fill out a Transgenic
Miscellaneous Service Form. Be advised that the training is free
but there is a charge if the facility performs the injection. It is
preferred that IV injections are scheduled for Friday afternoons to
avoid conflicts and risk to the facility animal room/procedures. If
the facility is to perform the IV injection, please inform Dr.
Ken Chen of the nature of the agent to be injected. Facility personnel
will go to the respective lab's desired injection site and perform the
service.
Q: Do you offer training in oviduct isolation or uterine dissection?
A: Yes the facility offers training in either at no charge.
Simply contact Dr. Ken Chen to schedule a
day for the training.
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