TRANSGENESIS IN MICE BY CYTOPLASMIC INJECTION OF POLYLYSINE DNA MIXTURES/

Pronuclear injection is currently the most often used method to make t ransgenic animals, but in some animal species it is temporally restric tive due to difficulty in visualizing pronuclei. However, the injectio n of construct DNA into the cytoplasm does not result in transgenesis. The production of transgenic mice by a cytoplasmic microinjection tec hnique of polylysine complexed DNA into pronuclear stage zygotes is de scribed. Transgenic mice were produced from cytoplasmic microinjection of mixtures of a 5.3 kb linearized DNA and poly-L-lysine (degree of p olymerization = 51). Effects on transgenic frequency of both the lysin e to phosphate ratio of polylysine to DNA and DNA concentration were s tudied. About 12.8% of the pups born from zygotes cytoplasmically micr oinjected with a polylysine/DNA mixture having a lysine to phosphate r atio (L:P) of 1:1 at a DNA concentration of 50 mu g ml(-1) were transg enic. The transgenic frequency for the pronuclear microinjection posit ive control of DNA alone was 21.7%. No transgenic pups were born from microinjection of DNA alone into the cytoplasm. Complexes of polylysin e/DNA were detected using agarose gel electrophoresis at the condition s which produced transgenic mice. The presence of polylysine with cons truct DNA altered the in vitro activities of restriction endonuclease and DNA ligase on the construct DNA. The production of transgenic anim als using DNA and polylysine in the absence of any other signal protei n suggests that a DNA/polylysine complex but not DNA alone can act as a substrate for transgenesis from the cytoplasm.