The Preparation and Analysis of DNA for Use in Transgenic Technology

The techniques of pronuclear and blastocyst injection are now routine procedures for making transgenic and gene-targeted mice, respectively. For many institutions, Core Facilities are the standard venue for producing such genetically modified animals for use in both basic and biomedical research. While the overall approach to microinjection has changed little over the years, the methodologies for the preparation of DNA for pronuclear injection and electroporation into embryonic stem (ES) cells has seen considerable improvement. Most significantly, procedures for DNA purification have been much simplified. In addition, rapid and cost efficient protocols are now available for genotyping of transgenic, gene-targeted, and mutant mice. In this chapter, we describe protocols that we employ to purify DNA for transgenic and gene-targeting work, as well as procedures for the preparation of DNA for both genotyping mice by PCR and by Southern blot analysis. The major goal behind the revision of these techniques has been to reduce the time and labor needed in order to obtain high quality DNA in sufficient quantities for specific procedures.