Purdue University Transgenic Mouse Core Facility Instruction Manual for Users
This manual has been designed to assist investigators in using the TMCF facility. It is broken down into three major sections as described below. Please remember that this manual is only a guide. If you require consultation of any kind please do not hesitate to contact the TMCF office directly.
Section I. General InformationThis section describes the core facility and its services in general terms. It also includes the price rates for 2012-2013.
IntroductionThe Purdue University Cancer Center Transgenic Mouse Core Facility (TMCF) is located in the Hansen Life Sciences building and serves as a centralized resource for the production of transgenic and gene-targeted mice for Purdue investigators. The Facility provides two basic services: 1) transgenic mouse generation and 2) embryonic stem cell targeting and blastocyst injection to generate gene altered mice. These two services are explained in more detail below. In addition, the TMCF can provide expert consultation concerning the design and execution of these experiments. The TMCF director, Judy Hallett, has extensive experience in both transgenic and knock-out experiments. This information packet is designed to provide the basic information required to initiate a project with the TMCF. However, if you find that you need further assistance please do not hesitate to contact the TMCF office.
The FacilityThe TMCF is located in the basement of the Hansen Life Sciences building and contains state of the art microinjection and cell culture facilities. In addition, the core has tissue sectioning equipment for histological analysis of embryos. Within the TMCF there is a fully functional barrier containment facility where all the animals are housed. All food and bedding are autoclaved and mice are housed in microisolator cages which are changed under laminar flow hoods. Access to this part of the TMCF is highly restricted and all personnel entering the barrier must wear full surgical gear.
Description of Services1) Transgenic Mouse Production
The transgenic mouse has assumed increasing importance as an experimental system
in recent years. The uses of this technology are numerous, but they include
studies of normal and altered gene function, analysis of elements responsible
for developmental and tissue-specific gene expression, and the production of
animal disease models. Transgenic mice are generated by injection of a recombinant
DNA construct directly into the male pronucleus of a fertilized egg. The injected
eggs are transferred to pseudopregnant females and the resultant pups are tested
for the presence of the transgene by Southern blotting of DNA from tail biopsies.
Multiple copies of the transgene integrate into a single, unique site usually
in a head to tail array. Therefore, each transgene positive founder will represent
an unique integration site which may affect transgene expression or regulation.
The Core will inject an individual transgene construct up to three times with
the goal of producing up to 15 potentially transgenic pups from each injection session. Tail DNA will then
be provided to the investigators for analysis. Once transgene positive pups
are identified, they will be given to the investigator and placed in the mouse
facility of their choice.
The ability to specifically alter the germline of mice using embryonic stem (ES) cell technology has truly revolutionized modern mammalian biology. In brief, gene targeting vectors are designed which, through homologous recombination, replace the wild-type allele of a given gene with a mutated form. The targeted ES cells are then implanted into day 3.5 blastocysts and transferred to pseudopregnant mothers. The resulting pups are chimeras between the ES cells (129/SvJ), which provide a brown coat color, and the C57Bl/6 blastocyst which has a black coat color. Animals with a high percentage brown coat color are bred and the resultant pups analyzed for the presence of the targeted allele. The core will electroporate a single gene targeting construct into ES cells and provide the investigator with DNA from at least 150 potentially targeted clones. Up to three positive clones will be injected into blastocysts and chimeric animals will be produced. Those with the highest percent chimerism will be transferred to the investigator's animal facility for breeding.
Service Charges 2012-2013Service:Cancer Center Member Rate/Non Member Rate
Transgenic production: $2,100/$5,320
Gene Targeting: *$7,010/$21,340
* Gene targeting experiments include ES cell targeting and selection, blast injection and chimeric pups.