Mouse Embryonic Stem Cell and
Transgenesis Core Laboratories
Services
DNA Injection: Upon receipt of the completed application form
and the DNA construct, proven by
documentation to be of high quality and suitable for microinjection, the core
will
proceed to clean the DNA sample and to inject a minimum of 200 fertilized
eggs (enough to generate 2-10 individual transgenic founder mice). Embryos
will be transferred into foster females and their pregnancy will be
monitored (the gestation period of the mouse is 19-20 days). Ten days after
birth of potential transgenic pups, the project scientist will be notified
of the number of pups. The core will carry out tail clipping and animal
tagging prior to weaning. Tail biopsies will be conveyed to the project
scientist for analysis by Southern blotting, PCR, or both. At this point,
the core will ask the project scientist to process the samples as quickly as possible (in
two weeks). If no result is received from PI there will be $1.00
charge for a cage per day. After weaning, the tagged pups will be
transferred to the care of the individual investigator who will be
responsible for subsequent maintenance.
Since cage costs are high and storage space is limited,
investigators should make certain that DNA screening procedures are working
prior to initiating transgenic work with the core. Investigators must
report the results of screening with documentation (gel photo, etc.) to the
Director. This information is necessary for core personnel to maintain
and improve their efficiency. If these studies (with adequate controls) fail
to demonstrate the existence of at least one transgenic animal, we will
reinject at least 200 eggs at no additional cost to the investigator. If,
however, no transgenic animal is detected after the second trial, a meeting
will be held between the scientist, the Core Director, and the
Consultants to discuss the DNA construct, the tail biopsy analysis, and
further plans to be agreed upon.
It is well understood that many factors can affect the
production efficiency of transgenic mice. Such factors include:
-
Number of eggs surviving the injection and developing to 2-cell stage;
this will determine the number of transfers into foster females. It is
important to have a DNA preparation pure of any contaminant and at the
appropriate concentration so that toxic effects to the eggs can be
avoided. Therefore we will clean the DNA samples after receiving it.
-
Successful pregnancies; although every transfer promises a certain number
of pups, that number may vary greatly due to embryo death in utero. This
lethality may be closely associated with the type of DNA construct used.
ES Cell Injection: Investigators are
encourages to submit a preliminary application form with the planned targeting before proceeding with
target construct preparation. Investigators are also encouraged to adhere to
the DNA preparation guide.Through this service,
the core will generate chimeric mice by injecting Embryonic Stem (ES)
cells containing site-specific genomic alterations (knockouts, knock-ins,
etc)) into normal blastocysts. ES cells will be injected into a minimum of 100 blastocysts.
The blastocysts will be transferred into the uterine horn of a 3-day
pseudopregnant female and allowed to go to term (17 days after embryo
transfer).
Upon weaning 3 weeks after birth, chimeric mice will be
separated and held until they reach sexual maturity (~6 weeks), then will be
mated with a C57B/6 female up to 5 litters.
Due to the fact that this approach is time-consuming,
it is recommended that certain guidelines be followed in order to minimize
loss of time and reduce risk factors. These guidelines include:
-
Before initiating the electroporation of targeted construct all the
procedural requirements that asked in the application must be met.
-
The targeting gene detection strategies must be determined before any
assigned electroporation day.
-
A photocopy of the recombinant ES cell line screening results (Southern
blot or PCR) should be submitted along with the chimeric mice request
form.
-
On the assigned day, the freshly harvested ES cell sample (free of feeder
cells) will be delivered to the microinjection suite to proceed with the
injection.
Embryo and Sperm Cryopreservation:
Freezing mouse embryos is a convenient way to preserve important mouse
lines for possible future use or to protect lines against loss due to
infection, lab accident, etc.
Embryo freezing could also result in substantial
reduction in housing cost and cage use. This newly added service is now
available to interested investigators. The service can be designed to suit
the needs of each investigator. However, a general scheme is outlined below.
A breeding pair of mice, or at least a young adult
male, will be needed to establish the small colony required to generate
enough embryos for each freezing set. A minimum of 400 healthy embryos
will be frozen per transgenic/knockout line that is hemizygous for the
transgene. In the case where the mouse line is homozygous for the transgene,
250 embryos will suffice. Several weeks after the completion of the freezing
process, a few embryo samples will be removed and processed through thawing
and monitoring development to assess their viability and ability to develop
to term. High
efficiency of embryo freezing will depend primarily on the reproductive
level of the breeding pair since low mating frequency and low percentage of
fertilization will lead to a prolonged time-consuming process.
For sperm cryopreservation, two adult mice preferably
12-16 weeks old should be submitted to the core after 2 to 3 weeks of
abstinence. Sperm samples will be collected from cauda
epididymis and will be frozen by way of vitrification. One week later a
single straw will be thawed to check the viability rate of spermatozoa.
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