Nanoinjection: pronuclear DNA delivery using a charged lance
- 780 Downloads
We present a non-fluidic pronuclear injection method using a silicon microchip “nanoinjector” composed of a microelectromechanical system with a solid, electrically conductive lance. Unlike microinjection which uses fluid delivery of DNA, nanoinjection electrically accumulates DNA on the lance, the DNA-coated lance is inserted into the pronucleus, and DNA is electrically released. We compared nanoinjection and microinjection side-by-side over the course of 4 days, injecting 1,013 eggs between the two groups. Nanoinjected zygotes had significantly higher rates of integration per injected embryo, with 6.2 % integration for nanoinjected embryos compared to 1.6 % integration for microinjected embryos. This advantage is explained by nanoinjected zygotes’ significantly higher viability in two stages of development: zygote progress to two-cell stage, and progress from two-cell stage embryos to birth. We observed that 77.6 % of nanoinjected zygotes proceeded to two-cell stage compared to 54.7 % of microinjected zygotes. Of the healthy two-cell stage embryos, 52.4 % from the nanoinjection group and 23.9 % from the microinjected group developed into pups. Structural advantages of the nanoinjector are likely to contribute to the high viability observed. For instance, because charge is used to retain and release DNA, extracellular fluid is not injected into the pronucleus and the cross-sectional area of the nanoinjection lance (0.06 µm2) is smaller than that of a microinjection pipette tip (0.78 µm2). According to results from the comparative nanoinjection versus microinjection study, we conclude that nanoinjection is a viable method of pronuclear DNA transfer which presents viability advantages over microinjection.
KeywordsNanoinjection Microinjection Transgenic DNA transfer MEMS
The authors would like to thank the students of the Brigham Young University Department of Mechanical Engineering Compliant Mechanisms Research Group (CMR) including SEM work by Gregory Teichert and Melanie Easter. We acknowledge the work of technician, Robert Rawle, and the students of the Brigham Young University Department of Molecular & Microbiology Burnett Research Laboratory. We also thank Phil Clair and Julie Tomlin of the University of Utah Transgenic and Gene Targeting Mouse Core for their excellent microinjection and surgical skills 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 Grants No. CMS-0428532 and No. CMMI-0800606. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation. Parts of this work have been presented at the 9th Transgenic Technology Meeting (abstract 42; March 2010) and the 10th Transgenic Technology Meeting (abstract #48; October 2011).
Online Resources 1 - Computer animation video of pronuclear nanoinjection. This video demonstrates the movement of the nanoinjector from its lowered position to an elevated position for injection. A close up view demonstrates the size of the lance in respect to the zygote and depicts the smooth motion of the lance entering and exiting the cell. Supplementary material 1 (WMV 8716 kb)
Online Resources 2 – Live video of pronuclear nanoinjection on a living mouse zygote. This video shows nanoinjection being performed on a living zygote. The zygote is held in position with a holding pipette and has been oriented to bring the pronuclei into the same plane as the lance. The appearance of the cell and the visibility of the pronuclei differ from images of typical inverted microscopy videos due to the use of an overhead camera for this video (refracted light and the curvature of the meniscus of fluid covering the nanoinjection chip causes this effect); therefore, the location of the pronuclei are indicated prior to nanoinjection with the lance. A notable feature of nanoinjection is the ease with which the lance penetrates the zona pellucida, the cell membrane, and the pronucleus, resulting in only very subtle inward depression of these membranes during penetration compared to what is typically seen with microinjection. Supplementary material 2 (WMV 13432 kb)
- Aten Q, Jensen B, Burnett S, Howell LL (2011) Electrostatic accumulation and release of DNA using a micromachined lance. J MEMS 20:1449–1461Google Scholar
- Audubert R, Mende S (1960) The principles of electrophoresis. Macmillan, New YorkGoogle Scholar
- Brown L, Cai T, DasGupta A (2001) Interval estimation for a binomail proportion. Stat Sci 16:101–133Google Scholar
- Everitt B (1992) The analysis of contigency tables. Chapman & Hall, New YorkGoogle Scholar
- Howell LL (2001) Compliant mechanisms. Wiley, New YorkGoogle Scholar
- Nagy A, Gertenstein M, Vintersten K, Behringer R (2003) Manipulating the mouse embryo: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
- Strowig T, Rongvaux A, Rathinam C, Takizawa H, Borsotti C, Philbrick W, Eynon EE, Manz MG, Flavell RA (2011) Transgenic expression of human signal regulatory protein alpha in Rag2−/−gamma(c)−/− mice improves engraftment of human hematopoietic cells in humanized mice. Proc Natl Acad Sci USA 108:13218–13223PubMedCrossRefGoogle Scholar