Transgene delivery via intracellular electroporetic nanoinjection
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Development of an effective cytoplasmic delivery technique has remained an elusive goal for decades despite the success of pronuclear microinjection. Cytoplasmic injections are faster and easier than pronuclear injection and do not require the pronuclei to be visible; yet previous attempts to develop cytoplasmic injection have met with limited success. In this work we report a cytoplasmic delivery method termed intracellular electroporetic nanoinjection (IEN). IEN is unique in that it manipulates transgenes using electrical forces. The microelectromechanical system (MEMS) uses electrostatic charge to physically pick up transgenes and place them in the cytoplasm. The transgenes are then propelled through the cytoplasm and electroporated into the pronuclei using electrical pulses. Standard electroporation of whole embryos has not resulted in transgenic animals, but the MEMS device allows localized electroporation to occur within the cytoplasm for transgene delivery from the cytoplasm to the pronucleus. In this report we describe the principles which allow localized electroporation of the pronuclei including: the location of mouse pronuclei between 21 and 28 h post-hCG treatment, modeling data predicting the voltages needed for localized electroporation of pronuclei, and data on electric-field-driven movement of transgenes. We further report results of an IEN versus microinjection comparative study in which IEN produced transgenic pups with viability, transgene integration, and expression rates statistically comparable to microinjection. The ability to perform injections without visualizing or puncturing the pronuclei will widely benefit transgenic research, and will be particularly advantageous for the production of transgenic animals with embryos exhibiting reduced pronuclear visibility.
KeywordsNanoinjection Microinjection Transgenic Electroporation Pronuclear migration DNA transfer
The authors thank the students of the Brigham Young University Department of Molecular & Microbiology Burnett Research Laboratory, the students of the Brigham Young University Department of Mechanical Engineering Compliant Mechanisms Research Group (CMR) and the University of Utah Transgenic and Gene Targeting Mouse Core for their contributions to this project. We also acknowledge our technicians Robert Rawle, Phil Clair, and Julie Tomlin for their continued assistance throughout this project. The authors recognize the funding support provided by Crocker Ventures, LLC and Nanoinjection Technologies, LLC. This material is based upon work supported in part by the National Science Foundation under Grant No. CMMI-0800606. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation.
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