Transgenic Animals with Various YAC Constructs: Preparation of 400 kb YAC DNA for Microinjection

Abstract

Abstract. The production of transgenic animals with YAC constructs is required for overcoming position effects on transgene expression. The preparation of YAC DNAs for microinjection is a critical factor in the production of transgenic animals carrying large YAC DNA inserts. We report a method for the preparation of 210-400 kb intact YAC DNAs for microinjection. We screened four YAC clones containing the α-lactalbumin gene, glutathione S-transferase alpha class gene, hepatocyte growth factor gene and casein gene family from a human YAC library, and analyzed the location of the genes in the YAC inserts to determine which fragmentation vectors should be used. YAC clones were transferred from their hosts to the CGY2516 yeast strain and fragmented to appropriate lengths to allow YAC copy number amplification. The YAC DNA was prepared and concentrated by filter centrifugation. Two-five ng/µl of intact purified YAC DNA was obtained. The YAC DNA was microinjected into rat embryos and transgenic rats were produced as efficiently as with conventional plasmid constructs.

Generation of Transgenic Rats with YACs and BACs: Preparation Procedures and Integrity on Microinjected DNA.

“…YAC DNA (yLALBA: 210 kb) containing the human α-lactalbumin gene was prepared according to the method described previously [7,8]. In brief, YAC DNA with or without restriction nuclease treatment was separated by pulsed field gel electrophoresis (PFGE, CHEF-DRII, Bio-Rad Hercules, CA) with 0.8% lowmelting temperature agarose (SeaPlaque GTG agarose, FMC BioProducts, Rockland, ME).…”