TRANSGENIC ANIMAL CORE (TAC)

Services

Pronuclear microinjection for the purpose of making transgenic animals is available only to researchers within the University of Missouri system and affiliated institutions for their basic research projects or as part of basic research collaborations with other academic institutions. Methods of pronuclear microinjection are patented under United States Patent No. 4,873,191, entitled: “Genetic Transformation of Zygotes,” assigned to Ohio University and licensed to DNX Transgenic Sciences.

DNA Preparation
The Transgenic Animal Core will work with the investigator to ensure that the DNA is of sufficient quality for microinjection. Dr. Natalia Karasseva is available to consult with investigators regarding construct design and DNA preparation. The purity of DNA sample is a critical step in successful generation of transgenic mice. For DNA purification the Transgenic Animal Core asks investigator to use QIAGEN Plasmid kit avoiding any ‘home-made’ protocols. Dr. Karasseva will perform excision of transgene from DNA construct and final purification of DNA insert prior to microinjection. Each investigator should submit at least 50 mg DNA construct, along with map indicating the restriction sites for transgene and the size of insert. (click here for DNA purification guidelines)

Production of Transgenic Mice by Pronuclear Injection
The Core will perform pronuclear injections, embryo transfer, house recipients during pregnancy and house litters until weaning age. At time of weaning, the core will obtain samples from all offspring and perform DNA preparation and initial screening for presence of transgene by PCR. To effectively screen offspring, the Investigator is requested to design and provide three sets of primers to generate DNA fragments from 300 to 500 bp long. All sets of primers have to be tested by PCR using 10 pg of DNA construct prior the submission. The Transgenic Animal Core will evaluate the sensitivity and specificity of designed primers. PCR primers can be ordered from any company of the Investigator choice. In past the Transgenic Animal Core had good experience with Integrated DNA Technologies Company www.idtdna.com, which provides reasonably priced, good quality and quick oligonucleotide synthesis service. Standard desalting has sufficient quality for purification of PCR primers.

Selection of Mouse Strain
The choice of mouse strain impacts several aspects of transgenic animal research. The first consideration is reproductive capacity. In general, outbred and hybrid strains have more rigorous reproductive performance. This results in a greater number of embryos to work with, an overall higher pregnancy rate after transfer, and the production of more offspring. Unfortunately, this strategy results in the transgene being expressed in various outbred backgrounds.

For some studies, the selection of an inbred mouse line will be important to the final project for which the transgenic animal is being produced. If the transgenic animal is produced on a mixed genetic background, the investigator will often need to backcross for many generations to establish an inbred line. Alternatively, the transgene may be introduced into the inbred line directly. The use of inbred animals as embryo donors reduces the overall efficiency of transgenic animal production, but may still be the appropriate strategy for the long term goals of the project.

The Core is currently working with the FVB mouse, which is an inbred animal with more moderate reproductive characteristics. If your project requires a specific animal strain, please consult with the Director of the core.

Health Monitoring
Additional health monitoring is performed on dams from litters with positive offspring before animals are provided to the investigator. At this time, the Core does not have adequate animal housing space to maintain or breed transgenic offspring. Positive animals will be provided to the investigator after initial screening when health monitoring is complete.

MU Animal Care and Use Committee (ACUC)
The Core has an ACUC protocol for the generation of transgenic animals. Each investigator is asked to submit a project specific ACUC protocol for review and approval prior to the creation of transgenic animals. (ACUC home)

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Services
Staff / contact information
Submission request
General timeline for pronuclear injections
MU Molecular Biology Program
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