Generation of transgenic rats with YACs and BACs: Preparation procedures and integrity of microinjected DNA

The aim of the present study was to investigate differences in the methods for preparing a large DNA fragment to be used for making transgenic rats fr om the standpoint of transgenic production efficiency and integrity of the introduced gene. In yeast artificial chromosome (YAC) transgenesis, three m ethods for preparing DNA for microinjection were compared: amplification of YAC in yeast (AMP), amplification of YAC in yeast and removal of the ampli fication element (AMP/RE), and no amplification of the YAC in yeast (AMP(-) ). Production efficiency per microinjected ovum with DNA by the AMP method was four times higher than that by the AMP/RE and AMP(-). Based on these re sults, we favor the AMP method in spite of the thymidine kinase gene-induce d male sterility. In bacterial artificial chromosome (BAC) transgenesis, li near DNA fragments for microinjection prepared by three kinds of purificati on procedures were compared: Not I digestion and CsCl gradient ultra-centri fugation (Prep.1), CsCl gradient ultra-centrifugation, Not I digestion, gel electrophoresis, and beta-agarase digestion (Prep. 2), and CsCl gradient u ltra-centrifugation, Not I digestion, pulse field gel electrophoresis, and beta-agarase digestion (Prep. 3). Although the efficiency of producing tran sgenic rats was similar with all these three DNA preparations, integration of the intact DNA fragment only occurred with the Prep. 3 procedure. We the refore favor the Prep. 3 method for preparing BAC DNA fragments. These resu lts indicate that the method used to prepare a large DNA fragment such as Y AC and BAC DNAs is important in order to produce transgenic rats with an in tact transgene.