in single embryos and live-born pups obtained by the co-injection procedures were 17.9 compared with 9.1 obtained by the injection of DNA alone. The results suggest that co-injection
of foreign genes with restriction enzyme may elevate the integration rate of foreign genes into host genomes. q 2000 Elsevier Science B.V. All rights reserved.
Keywords: Co-injection; Transgenic mice; Restriction enzyme; PCR; Transgenesis
1. Introduction
Microinjection of foreign DNA into the pronuclei of fertilized eggs has been extensively employed for the production of transgenic animals; however, this method for
gene transfer requires an extremely specialized technique. In addition to the technical obstacle, the rate of successful incorporation of the exogenously introduced genes into
the host genome has been intolerably low, particularly in farm animals. Approximately
Ž 5 of mouse eggs microinjected with foreign DNA result in transgenic mice Brinster et
. Ž
al., 1985 , while the efficiency is 0–5 in swine, and less than 1 in sheep Pursel et .
Ž .
al., 1989 and cattle Krimpenfort et al., 1991; Hill et al., 1992 . Several attempts have been made to simplify microinjection technique and improve
Ž .
the efficiency of integration of foreign DNA. Page et al. 1995 attempted to produce transgenic mice with the cytoplasmic injection of DNA mixed with polylysine, which
Ž has been used to transfect some mammalian cell lines Felgner et al., 1987; Behr et al.,
. 1989 . They reported that about 12.8 of the pups born from zygotes cytoplasmically
microinjected with a polylysinerDNA mixture were transgenic, whereas no transgenic pups were born from the microinjection of DNA alone into the cytoplasm. It has
generally been believed that the opportunity for foreign DNA integrated into the genome occurs only when host DNA molecules are cleaved as a result of the stimulation caused
Ž .
by the microinjection and rejoined during the repair Brinster et al., 1985 . Ž
. Phillips and Morgan 1994 induced the DNA double-strand breaks in the endoge-
Ž .
nous gene in cultured Chinese hamster ovary CHO
cells by electroporating the restriction endonuclease into the cells, and they found that blunt-end DNA double-strand
Ž .
breaks can induce illegitimate nonhomologous recombination in mammalian chromo- Ž
. somes. Brenneman et al. 1996 demonstrated that the intrachromosomal homologous
recombination in human cells was stimulated by the introduction of foreign DNA together with site-specific endonuclease. Thus, it appears that the introduction of certain
restriction endonuclease into cultured mammalian cells may significantly stimulate the DNA recombination between the foreign DNA and the host genomic DNA. Although
the precise details of the mechanisms of integration are still unclear, it has been proposed that the DNA integration processes in fertilized eggs and cultured cells have
Ž .
the same molecular basis Bishop and Smith, 1989 . In this work, we have attempted to investigate whether or not co-injection of foreign DNA constructs with bacterial
restriction endonuclease into the pronucleus of mouse zygotes could improve the integration frequencies of foreign DNA into host genomes, which in turn could result in
a higher production rate of transgenic animals.
2. Materials and methods
