Co-injection of restriction enzyme with foreign DNA into the pronucleus for elevating production efficiencies of transgenic animals

The microinjection method for production of transgenic farm animals require s specialized techniques and results in intolerably low production efficien cies. We investigated whether or not co-injection of foreign DNA constructs with restriction endonuclease into the pronucleus of mouse zygotes would i mprove the integration frequencies of foreign DNA into the host genome. Two kinds of DNA constructs that have no EcoRI site in their sequences were us ed for co-microinjection. With reference to the results of experiments in w hich EcoRI alone was injected at various amounts varying from 10(-9) to 10( -5) U/nucleus, the amount of 5 x 10(-8) U/nucleus that showed survival rate of 60.6% was used for the co-injection with DNA. Successful transgenesis o f co-injected embryos was identified by DpnI-Bal31 digestion method for sin gle embryos and by PCR method for pups born, respectively. The overall effi ciency for the integration of foreign DNA in single embryos and live-born p ups obtained by the co-injection procedures were 17.9% compared with 9.1% o btained by the injection of DNA alone. The results suggest that co-injectio n of foreign genes with restriction enzyme may elevate the integration rate of foreign genes into host genomes. (C) 2000 Elsevier Science B.V. All rig hts reserved.