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Transgenic mice are constructed by injecting cloned DNA into
fertilized mouse eggs; those eggs that survive are then implanted in
foster females to develop to term. The gestation period of the mouse is
19-21 days. Pups are ready for weaning at 3-4 weeks of age, and reach
sexual maturity at 6 weeks (females) to 8 weeks (males). The minimum
elapsed time between injection of the construct and readiness for
breeding of the transgenic founders is 9 to 11 weeks. For most
experimental purposes researchers will want to use offspring of a
transgenic animal, rather than the founder animal itself.
Several factors can affect the production of transgenic mice. For
example, the purity of the DNA construct to be injected is very
important to avoid toxic effects to the eggs, and the DNA must be at
the proper concentration. The DNA construct must be purified away from
vector sequences. Also, some expression constructs (e.g.
beta-actin-MyoD) may have activities that are lethal for the transgenic
embryos. Another caveat is that if transgene integration occurs after
the first round of DNA replication in the single fertilized mouse egg,
the mouse will be mosaic for the transgene, i.e. only a subset of cells
will carry the transgene, and if the germ cells don't carry the
transgene, it will not transmit to the offspring. We will make every
reasonable effort to make transgenic mice with your construct, but no
facility can guarantee success with every construct.
The GTTF will isolate the transgene DNA from the DNA constructs
provided by the investigator, then microinject this into approximately
300 fertilized eggs, transfer the embryos into foster females and
monitor the pregnancies. The GTTF will perform the tail biopsies and
tag the pups, then transfer the pups and biopsies to the investigator.
The investigator will perform DNA isolation from the tail clips, and
analyze the DNA by PCR and/or Southern blotting in a timely manner.
Pups, including potential transgenic founder animals, will be
transferred to the care of the investigator upon weaning (3 weeks of
age), who will arrange appropriate Vivarium housing and breeding for
them outside of our facility.
If the studies on tail biopsy DNA (with adequate controls) fail to
demonstrate the presence of at least three transgenic animals, we will
reinject the construct at no additional cost to the investigator. If no
transgenic animal is detected after the second set of injections, we
will hold a meeting to discuss further plans, such as re-purification
of the DNA construct, or repeat injections and analysis of potential
transgenic embryos if the DNA construct may result in embryonic
lethality.
The investigator should provide a high purity DNA construct, the
GTTF will further purify the transgene away from the vector. The
investigator must purify DNA from the tail biopsies and analyze the
samples to determine which mice carry the transgene, in a timely
manner. The pups, including the transgenic founder mice, will be
transferred out of the facility to the care of the investigator upon
weaning.
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Transgenes should include promoter, enhancer, gene to be expressed,
splice donor and acceptor and intron sequences, and
termination/polyadenylation sequences. Transgenes must be excised from
the bacterial plasmid sequences in order to be expressed in mice.
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