title: DNA preparation method can influence outcome of transgenic animal experiments.
abstract: In our continuing quest to improve the efficiency of producing transgenic animals, we have compared the influence of two transgene purification techniques on the efficiency of creating transgenic sheep and mice. Three hundred eighty-seven sheep zygotes and 2,737 mouse zygotes were microinjected with one of four transgenes. Transgenes were isolated from plasmid sequences either by agarose gel electrophoresis followed by gel extraction or by a single step sodium chloride gradient fractionation technique. Four transgenic sheep and 61 transgenic mice were produced. Both sheep and mice embryos responded similarly to transgene preparation methods. Overall, pregnancy rate was higher for recipients that received embryos injected with NaCl purified DNA (mean +/- SEM: 64 +/- 7% vs. 38 +/- 7%). Furthermore, offspring per zygote transferred (NaCl, 22 +/- 3% vs. Gel, 12 +/- 3%) and transgenics born per zygote transferred (NaCl, 3.9 +/- 0.6% vs. Gel, 1.5 +/- 0.6%) were higher when the NaCl purified DNA was used. However, the proportion of offspring born that were identified as transgenic did not differ between transgene purification methods. Transgenes responded differently to methods of preparation. One of the four genes yielded a significantly higher proportion of transgenics when the transgene was prepared by NaCl purification. These data suggest that on average the NaCl gradient purification technique results in a higher embryo survival rate to term for both sheep and mice, but the technique has no influence on rate of transgene integration.
authors.affiliation: Gene Evaluation and Mapping Laboratory, USDA, Beltsville, MD 20705, USA. bobwall@lpsi.barc.usda.gov
authors.forename: R J
authors.initials: RJ
authors.lastname: Wall
authors.forename: R K
authors.initials: RK
authors.lastname: Paleyanda
authors.forename: J A
authors.initials: JA
authors.lastname: Foster
authors.forename: A
authors.initials: A
authors.lastname: Powell
authors.forename: C
authors.initials: C
authors.lastname: Rexroad
authors.forename: H
authors.initials: H
authors.lastname: Lubon
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D004247:DNA / Q000302:isolation & purification*; D004624:Embryo Transfer / Q000662:veterinary; D005260:Female; D051379:Mice; D008822:Mice, Transgenic; D008845:Microinjections / Q000662:veterinary; D011247:Pregnancy; D012756:Sheep; D019076:Transgenes
chemical_list: D004247:DNA