University of Hawaii - Transgenic Core

Transgenic subjects carrying new or novel genes are created by microinjection of DNA into the pronuclei of fertilized eggs. Knock-out lacking specific genes of interest are created by homologous recombination in embryonic stem cells followed by injection into blastocysts to create chimeric subjects. Highly experienced personnel produce transgenic and knock-out subjects for UH investigators at very reasonable cost and with very short lead times.

Services
The Basic Services of the Transgenic Core Facility are:

DNA Injections
Embryonic Stem Cell Injection
Embryo Freezing
In addition to the services the Transgenic Core Facility provides, the following techniques are available to the research community:

Embryo Culture
Embryo Transfer
Embryo Micromanipulation
Mouse In Vitro Fertilization
Mouse Cloning Services
Pronuclear Microinjection Services

The purity of the microinjected DNA fragment is critical for successful production of transgenic founder mice. DNA impurities in the form of bacterial endotoxins or organic contaminants can decrease integration efficiency of the fragment or the viability of the microinjected embryos. The following conditions must be met by the investigator for submission of plasmid DNA:

A map of the plasmid, delineating the insert and relevant restriction sites.
Plasmid DNA is purified preferably on a cesium chloride gradient, or using of the Qiagen Plasmid Maxi kit.
The presence of prokaryotic sequences interferes with transgene expression. Thus the insert to be injected must be flanked by restriction site(s) that allow removal of vector sequences. The purity of the DNA preparation is to be documented with a gel photograph. The plasmid should be digested with the enzymes(s) used to release the insert from vector sequences, as well as two enzymes that cut within the insert and produce fragments of predicted size.
The Facility also notes that a transgene construct prepared from genomic DNA is preferable to one prepared from cDNA. Although there are reports of cDNA expression in the literature, the presence of introns increases the likelihood of transgene expression.

Once all of the above criteria are met, please submit a completed electronic application, DNA construct, and supporting documentation. Users will be scheduled for microinjection services on a first-come, first-serve basis. Notification of the scheduled microinjection date will be sent via e-mail within 10 working days of the receipt of all application materials.