title: Transgenesis in mice by cytoplasmic injection of polylysine/DNA mixtures.
abstract: Pronuclear injection is currently the most often used method to make transgenic animals, but in some animal species it is temporally restrictive due to difficulty in visualizing pronuclei. However, the injection of construct DNA into the cytoplasm does not result in transgenesis. The production of transgenic mice by a cytoplasmic microinjection technique of polylysine complexed DNA into pronuclear stage zygotes is described. Transgenic mice were produced from cytoplasmic microinjection of mixtures of a 5.3 kb linearized DNA and poly-L-lysine (degree of polymerization = 51). Effects on transgenic frequency of both the lysine to phosphate ratio of polylysine to DNA and DNA concentration were studied. About 12.8% of the pups born from zygotes cytoplasmically microinjected with a polylysine/DNA mixture having a lysine to phosphate ratio (L:P) of 1:1 at a DNA concentration of 50 micrograms ml-1 were transgenic. The transgenic frequency for the pronuclear microinjection positive control of DNA alone was 21.7%. No transgenic pups were born from microinjection of DNA alone into the cytoplasm. Complexes of polylysine/DNA were detected using agarose gel electrophoresis at the conditions which produced transgenic mice. The presence of polylysine with construct DNA altered the in vitro activities of restriction endonuclease and DNA ligase on the construct DNA. The production of transgenic animals using DNA and polylysine in the absence of any other signal protein suggests that a DNA/polylysine complex but not DNA alone can act as a substrate for transgenesis from the cytoplasm.
authors.affiliation: Department of Chemical Engineering, Virginia Polytechnic Institute and State University, Blacksburg 24061, USA.
authors.forename: R L
authors.initials: RL
authors.lastname: Page
authors.forename: S P
authors.initials: SP
authors.lastname: Butler
authors.forename: A
authors.initials: A
authors.lastname: Subramanian
authors.forename: F C
authors.initials: FC
authors.lastname: Gwazdauskas
authors.forename: J L
authors.initials: JL
authors.lastname: Johnson
authors.forename: W H
authors.initials: WH
authors.lastname: Velander
mesh_terms: D000818:Animals; D003593:Cytoplasm; D011088:DNA Ligases; D004274:DNA, Recombinant / Q000032:analysis / Q000378:metabolism; D015245:Deoxyribonuclease BamHI; D005260:Female; D018014:Gene Transfer Techniques; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D008845:Microinjections / Q000379:methods*; D008892:Milk; D011107:Polylysine / Q000378:metabolism; D011486:Protein C / Q000096:biosynthesis / Q000235:genetics; D011993:Recombinant Fusion Proteins; D019076:Transgenes / Q000235:genetics*; D015053:Zygote
chemical_list: D004274:DNA, Recombinant; D011486:Protein C; D011993:Recombinant Fusion Proteins; D011107:Polylysine; D015245:Deoxyribonuclease BamHI; D011088:DNA Ligases