Procedures
for the Construction of a Transgenic Mouse Strain at JHU
The purpose of the School of Medicine Transgenic Mouse Core Laboratory (TCL) is
to make genetically modified mice for Johns Hopkins University researchers.
1.
Eligibility.
Approval of the Institutional Animal Care and Use Committee (IACUC) is needed
to breed, house and perform experiments with mice at JHU. Forms for protocol submission to the
IACUC are available from Comparative Medicine (955-3273). Do not include a description
of the procedures involved in the production of transgenic mice since that is
covered by the TCL protocol. A
current protocol number, representing IACUC approval is required on the DNA
Submission Form.
2. Priority. DNA constructs will be
injected for JHU investigators on a first come, first served basis with the
caveat that an investigator may only have 2 constructs in the queue at any
time. If the investigator submits
more than 2, the additional constructs will be held off the queue until one of
the first 2 is started. Only then
will an additional construct be added to the queue.
3. Requirements for admission to the injection queue. In order to have DNA
injected, investigators must comply with the requirements listed below:
a)
Submit
5 ug of clean, linearized DNA, diluted to 100ng/ul in T low E (10 mM TRIS, 0.1
mM EDTA), to be provided by the TCL. The cleanliness of the DNA submitted is
extremely important. Poorly
prepared DNA will greatly reduce the survival of the injected embryos leading
to few or no pups being born. Several protocols are available on the Core's website (DNA preparation for Microinjection). Additionally, incorrectly quantified
DNA preps can lead to either very poor integration rates (if there is less DNA
than stated), or greatly reduce the survival of the injected embryos leading to
few or no pups being born (if the DNA concentration is greater than
stated). The submitted construct
must be accompanied by a photograph of the DNA in an agarose gel with
appropriate size markers. We will attempt to work with smaller amounts of DNA
if necessary, but investigators are advised that 5 ug is the optimal amount.
b)
Present
a completed TCL DNA
Submission Form including a signed M&S form. The fee for injection of
one construct is $3500 for the standard strain (B6SJLF1) and $4500 + mouse
costs for nonstandard strains or for BAC injections.
c)
Have
a proven screening protocol developed. See part 5 for re-injection requirements.
4.
Responsibilities of the TCL: The Transgenic Core will
inject enough embryos to implant ~25 embryos into each of 10 recipient
females. This usually requires the
injection of 400-600 embryos over 2-4 days. This level of injection will usually result in 20-40 pups
being born. Approximately 10% of
pups born tend to be transgenic. The birth and transgenic rates are very dependent on the quality of the
DNA preparation and the accuracy of the concentration determination. If a greater than average amount of
embryo death is occurring, we will ask the researcher to re-prepare the
DNA. Poorly prepared DNA is
thought to be the most common reason for poor numbers of pups being born in a
well established microinjection lab.
5.
Investigators are responsible for identifying transgenic pups and notifying the
TCL of the results.
The TCL will attempt to transfer the Moms and pups to the investigator when the
pups are 3-4 weeks of age. It will
be the responsibility of the investigator's laboratory to wean (if not already
done) and screen the pups for the presence of the transgene. If a round of injections fails to
produce at least 2 founders, the TCL will re-inject the same construct one time. In order to be eligible for re-injection,
the animals must be screened and a re-injection request made within 6 weeks of
the transfer of the pups to the investigatorís animal colony. In order to re-inject, the person
making the request must be able to demonstrate that the screening protocol is
able to identify transgenic founders. Positive controls should include multiple dilutions of plasmid into a
wild type mouse DNA prep (at the same concentration of total DNA as the
potential founder preps), down to a plasmid concentration of 10fg/ul.
6. Maintaining
virus antibody-free mice. The TCL and Animal Services are following procedures to
maintain disease-free animals. Our
lab follows an extensive sentinel screening program in which every rack is
screened monthly. Additionally,
every litter of pups is tested for MHV, Helicobacter sp. and pinworms prior to
being transferred. Currently our
mice can be shipped to any animal facility at Hopkins directly except for CRB. Mice going to CRB must first be shipped
to BRB for additional monitoring.
Effective
12/1/04 CEH