Assessment of DNA Expression Following Cytoplasmic Microinjection of Condensed DNA into Murine Embryos Using Electropulsation
C.A. Schmotzer, S.P. Butler, R.E. Pearson, W.H. Velander and F.C. Gwazdauskas
Cytoplasmic injection of DNA has been an inefficient method of producing transgenic animals. However, the benefit of this method to embryo viability because of decreased manipulation warrants further investigation of this technique. The objective of this study was to evaluate electroporation as a method of facilitating gene integration into murine embryos following cytoplasmic injection of linear and condensed DNA constructs and to assess the effects of these techniques on subsequent embryo development. Non-manipulated control embryos had the highest d 4 development score (5.57 ± 0.11), while manipulated control embryos had a significantly (P < 0.01) lower developmental score (4.6 ± 0.18). Injecting DNA verses only inserting the injection needle had a significant negative impact on embryo development (2.42 + 0.12 verses 3.63 + 0.12; P < 0.01). Similarly, a 250 V electropulse reduced development compared to no electroporation (2.22 + 0.12 verses 3.43 + 0.12, P < 0.01). The DNA construct form (linear or condensed) had no impact (P > 0.05) on embryo development (2.83 + 0.19 verses 2.85 + 0.19). Zygotes injected with the condensed and linear DNA constructs had 0.38% of the embryos fluorescing at d 4 of in vitro culture. When DNA injection was followed by electropulsation, 0.36% of zygotes injected with condensed DNA and 4.64% of zygotes injected with linear DNA exhibited fluorescence. These data suggest that electropulsation of embryos following injection of linear DNA may improve integration and protein expression following cytoplasmic injection and may improve overall integration efficiency, since no mosaic embryos were found in this experiment. However, these procedures decrease development score dramatically.