Note:
It is required that the requesting investigator have an approved
IACUC protocol before submission of a request to the Mouse Transgenic
Facility.
*Any experiments involving the injection of cell lines provided by
the requesting investigator require documentation of MAP testing indicating
no detection of pathogens.
Pronuclear
Injections for Transgenic Embryo/Strain Generation
The user
submits a Pronuclear Injection Request Form and provides DNA for microinjection.
When submitting
DNA for pronuclear injection, please use this DNA
purification protocol. As the
quality of the DNA is critical for optimal success, extreme care must
be taken in the preparation of the DNA, and the core will request documentation
of the purity and quantitation of the purified sample. Microinjection
of DNA is performed routinely into FVB/N mouse embryos, although special
arrangements can be made for other strains, with the requesting investigator
covering additional mouse costs. Injected eggs are transferred
into a pseudopregnant recipient, and resultant pups are tagged and
tail biopsied for the detection of the transgene by the investigator.
Animals are housed within the core colony until genotyping is completed. The
founder animals are then transferred to the investigator.
Cost
for pronuclear injections for in-house MMCRI investigators is $400.00/injection
day. Two days of injections are recommended for each construct
to insure enough founders are produced. Approximately 100 eggs are
injected each day. In our experience, this leads to a minimum
to three to up to 20 founder lines generated.
*The
core facility is now accepting transgenic requests from investigators
outside MMCRI for $2,800.00 per construct, yielding a minimum of
three founder lines, assuming receipt of high quality DNA. Contact
Lucy Liaw to make arrangements (liawl@mmc.org).
Embryonic
Stem Cell Injections for Chimeric Mice
The investigator
submits an ES Cell Injection Request Form and provides ES cells that
have been tested and are negative for mycoplasma and mouse antibody
production (MAP) against murine viral pathogens. ES cells are injected
into C57BL/6 blastocysts. Each investigator should submit at
least 2 independent targeted clones to increase the chance of chimeric
mice being produced.
The cost
for ES cell injection for in-house MMCRI investigators is $800.00/clone.
Two days of injections are recommended, with approximately 50 blastocyst
injected.
Cryopreservation
Program
The
Transgenic Core is now offering embryo and sperm cryopreservation
for your mouse lines. Please read this description of both procedures
to help determine which option is best for you. Contact Anne Harrington
with any questions (harria@mmc.org).
Embryo
Cryopreservation
If you would like to maintain a line as a homozygote or your line
is on an inbred background, this is the best option for you.
The line can be frozen two ways. First, you supply the core with
10 males that are fertile and capable of producing live offspring.
Wildtype females will be ordered and used as egg donors to produce
2 cell embryos which will then be frozen. A sample straw will be
thawed to verify viability. The Investigator is responsible for supplying
proven young male studs, the embryo donors and covering per diem
housing costs. The second option is to supply the core with 10 homozygous
males and at least 30 (10/week) homozygous female egg donors. The
females must be 3-4 weeks of age or 8-15 weeks of age. The per diem charge
is applied.
Sperm Cryopreservation
This is the most cost effective option, however it has its limitations.
The core will collect sperm from your males and freeze down 20 straws
per male. Recovery of live mice from frozen mouse sperm is strain
dependent. I recommend that you order an in vitro fertilization (IVF)
service to test the sperm for their ability to generate viable pups.
IVF results vary according to genetic background and the quality
of individual males used for IVF. Thus we cannot offer a guarantee
that any given IVF procedure will produce large number of pups.
Cryopreservation Costs
Embryo Freeze (one straw will be thawed and cultured to blastocyst
stage)
The requesting investigator is responsible for mouse and per diem
housing costs. $400.00/session
Embryo Recovery*
$100.00/recipient + per diem housing cost
Sperm Freeze
$100.00/male
In vitro fertilization
The requesting investigator is responsible for mouse and per diem
housing costs.
$200.00/session
*vitrified embryos will not be accepted from MMRRC-UCD M
References:
Thornton C.E., Brown S.D.M, Glenister P.H. (1999). Large numbers of
mice established by in vitro fertilization with cryopreserved spermatozoa:
implications and applications for genetic resource banks, mutagenesis
screens, and mouse backcrosses. Mammalian Genome 10: 987-992.
G.Charles
Ostermeier, Michael V, Wiles, Jane S. Farley, Robert Taft (2008). Conserving,
Distributing and Managing Genetically Modified Mouse Lines by Sperm
Cryopreservation. PLos One 3:1-8
Re-derivation
by Oviduct Transfer of Zygotes
The investigator
submits a Re-derivation Request Form. All mice that enter MMCRI
from non-approved vendors MUST be re-derived and serology tested before
housing within the barrier facility.
The cost
for re-derivation is $400.00/line. Core staff will assist in
determining requirement of mice for each line. The core will cover
the cost of importation and re-derivation of all shared lines.
