A Comparative Study on the Integration of Exogenous DNA into Mouse, Rat, Rabbit, and Pig Genomes.

Abstract

Transgenic mammals, from small laboratory rodents to domestic animals, have been successfully produced to date, but their production efficiency within or across species has been variable. This is probably due to the differences in the type of injected DNA and/ or technical procedures employed in each laboratory, as well as the reproductive characteristics of the species. Here we report the direct comparison of the efficiencies of producing transgenic mice, rats, rabbits and pigs by one technician using a fusion gene composed of the bovine αS 1 -casein promoter and human growth hormone (hGH) gene. Before the fusion gene was injected into the zygotes, high magnitude centrifugation to visualize the pronuclei was necessary for all of the pig zygotes and one-third of the rabbit zygotes, but not for mouse and rat zygotes. Post-injection survival of the mouse zygotes (67.1%) was lower than those of the rat, rabbit and pig zygotes (89.6 to 100%). The volume change of the pronucleus following DNA injection was the lowest in mice (50% increase), moderate in rabbits (148% increase), and the most prominent in rats (238% increase). The data from only 1 pig zygote indicated a 22% increase in the pronucleus volume by DNA injection. The PCR analyses of the tail DNA of new born offspring indicated that 0.8% (4/ 493), 4.8% (22/463), 0.8% (3/367) and 0.9% (2/221) of the injected eggs in mice, rats, rabbits and pigs, respectively, developed into transgenic offspring. Some of the founder animals in all four species expressed the transgene in the mammary gland which was confirmed in hGH mRNA by RT-PCR and/or hGH peptide in Witch's milk with ELISA. These results suggest that the maximum volume of DNA solution injectable into the pronucleus is a possible factor explaining the species differences in the production of transgenic animals.

Effects of cryopreservation of pronuclear‐stage rabbit zygotes on the morphological survival, blastocyst formation, and full‐term development after DNA microinjection

“…Nevertheless to our knowledge, the 18 offspring obtained in the present study were the ®rst rabbits to develop from cryopreserved pronuclearstage zygotes. The overall ef®ciency of producing transgenic rabbits by DNA injection of fresh zygote was 0.7% in the present study, within the range of 0.3 to 2.5% reported to date (Snyder et al, 1995;Spieker-Polet et al, 1995;Aigner et al, 1996;Hirabayashi et al, 2000bHirabayashi et al, , 2001). Although we were unable to produce transgenic rabbits from cryopreserved pronuclear-stage zygotes in the present study, our results do not necessarily lead to the conclusion that cryopreserved pronuclear-stage rabbit zygotes will not be useful for production of transgenic rabbits by DNA microinjection.…”

“…The content of cytoplasmic lipid droplets in rabbit zygotes may be similar to those in mouse and rat zygotes, because pronuclei can be visualized in most pronuclear-stage rabbit zygotes even without centrifugation (Hirabayashi et al, 2000b), as is the case for these rodent zygotes (Hirabayashi et al, 2001). On the other hand, the diameter of rabbit zygotes (130±150 mm) is much larger than those of the rodents (70±90 mm), and is close to those of large domestic species such as cattle, pig, and sheep.…”

Expression of Porcine FSH.BETA. Subunit Promoter-driven Herpes Simplex Virus Thymidine Kinase Gene in Transgenic Rats

“…Essentially, generation of transgenic rats was accomplished using F344 rats by a method reported previously [16,17]. Since the transgenic rats produced showed male infertility, heterozygote offspring were obtained by crossing transgenic female rats with normal male rats.…”

Pig transgenesis by Sleeping Beauty DNA transposition

“…Transgenesis in pigs has previously been achieved by transferring the transgene to oocytes or early embryos by pronuclear injection of foreign DNA (Hammer et al 1985;Hirabayashi et al 2001;Nottle et al 2001), transduction with retro-and lentiviral vectors injected into the perivitelline space of the oocyte (Cabot et al 2001;Hofmann et al 2003;Whitelaw et al 2004) or, alternatively, by sperm-mediated gene transfer (Naruse et al 2005;Webster et al 2005). With the successful cloning of animals by somatic cell nuclear transfer (SCNT), it is now possible to produce transgenic pigs from genetically engineered somatic donor cells.…”