of Transgenic Mice or Rats
introduction of foreign DNA (transgenes) into the genome of laboratory
animals allows studies of gene expression and protein function within
live animals. The mouse is the species of choice for most investigators
due to their relatively low cost and the extensive research that has been
done in murine models in the past resulting in increased knowledge of
their genetics and physiology in various strains. In some cases,
however, investigators prefer to use rats due to factors such as physiological
and pathological similarities to humans or increased size. The
TAF offers the opportunity to produce transgenic mice and rats.
Essentially the steps necessary
for the production of transgenic mice/rats are:
- construction of a transgenic vector containing a suitable promoter,
a genomic clone or DNA and intron fragment, and polyadenylation
- isolation of the transgenic fragment free from prokaryotic vector
- purification of the transgene for microinjection
- microinjection of the transgene into the pronuclei of fertilized ova
to generate mice/rats containing copies of that transgene integrated
randomly in their genome
Prior to the start of pronuclear
microinjections, the investigator will need to meet with the TAF staff.
This consultation will enable us to determine the specific needs
of the investigator, review the design of the transgenic vector, and discuss
fees for various services. Please contact Kathy Krentz (firstname.lastname@example.org) or (608) 890-3785 to schedule an initial meeting.
Guidelines for Transgene Vector Design
recommend incorporating the following features in your transgenic vector.
with a known cellular expression profile
- a genomic
clone or cDNA and intron fragment containing splice donor and receptor
- a fragment
containing poly adenylation addition sequences
characterized restriction sites that will allow the isolation of a transgenic
fragment free from prokaryotic vector sequences
Purification of Transgene for Microinjection
purified DNA is essential for successful transgenic production. Particles
within the DNA could easily clog the microinjection needle and/or interfere
with integration of the DNA. Our facility will purify your digested DNA.
also has experience microinjecting BAC and PAC DNA. However, isolation
methods are different. Please contact us for the latest isolation procedure.
Microinjection of DNA into Oocytes
a billing request form
an IACUC approved animal
digested DNA along with a gel picture showing complete digestion and band to extract.
TAF will purify, verify the fragment size and dilute
the DNA for microinjection
a map of your transgene and vector
TAF routinely microinjects transgenes
into the pronucleus of FVB/n and C57BL/6 one-cell embryos. Other strains
will be considered only after meeting with Ms. Krentz to discuss the project.
The investigator may incur additional costs for the use of other strains
as embryo donors if costs of materials and time required to complete the
project are greater than for our standard strains.
Following the microinjections,
the embryos are transferred into the oviducts of pseudopregnant recipients.
Pups should be born 19 days later and weaned 3 weeks after birth. Investigators
are provided with tail DNA samples from each potential founder and have
three weeks to complete genotyping the mice. We recommend PCR analysis as the preferred method of genotyping. The investigator must provide a copy of the genotyping results
to the TAF staff. The TAF then will breed up to three founders when they
reach breeding age to wild-type mates to produce one F 1 litter from each
founder. The founder and litter are transferred to the investigator when
the progeny are three weeks old. Any remaining founders will be shipped
without breeding to the investigator
TAF routinely microinjects transgenes
into the pronucleus of Sprague-Dawley (outbred) one-cell embryos and has
also successfully produced transgenics on an F344 (inbred) background.
Other strains will be considered only after meeting with Ms. Krentz to
discuss project. The investigator may incur additional costs for
the use of other strains as embryo donors if costs of materials and time
required to complete the project are greater than for our standard strains.
Following the microinjections, the embryos are transferred into
the oviducts of pseudopregnant recipients. Pups should be born 22 days
later and weaned 3 weeks after birth.
At weaning, investigators are
provided with tail DNA samples from potential founders and have three
weeks to complete the genotyping. We recommend PCR analysis
as the preferred method of genotyping. The investigator must provide a copy of the genotyping results
to TAF staff. We will then breed the first three founders when
they reach breeding age to wild-type mates to produce one F1
litter from each founder. Any remaining founders will be shipped
to the investigator for breeding.