Transgenic Research

, Volume 21, Issue 6, pp 1279–1290 | Cite as

Nanoinjection: pronuclear DNA delivery using a charged lance

  • Quentin T. Aten
  • Brian D. Jensen
  • Susan Tamowski
  • Aubrey M. Wilson
  • Larry L. Howell
  • Sandra H. Burnett
Original Paper

Abstract

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.

Keywords

Nanoinjection Microinjection Transgenic DNA transfer MEMS 

Supplementary material

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)

11248_2012_9610_MOESM3_ESM.eps (89 kb)
Online Resources 3 – In vitro data confirms that nanoinjection maintains zygote viability compared to untreated zygotes. Results from the in vivo study did not indicate statistical difference between nanoinjected and untreated zygotes able to proceed to the 2-cell stage (Fig 5B). This Online Resources figure presents data from numerous in vitro studies as a second data set to confirm that the viability to the 2-cell stage is maintained in nanoinjected cells. We felt that this in vitro data was valuable to confirm in vivo work presented in the main body of this research for two reasons. First, the in vivo study only included 31 zygotes in the untreated controls compared to 371 in the nanoinjection group, but for in vitro studies, we collected data over more weeks, which includes more zygotes. A higher number in the untreated controls of the in vitro studies offers greater weight to the statistical interpretation, as is apparent when one compares the confidence intervals in Fig 5B to those shown in this figure. Second, we had access to a different strain of mouse for in vitro data (CD1 outbred) compared to in vivo data (C57Bl/6 J × CBA/J F1), yet the results demonstrate the ability of the nanoinjection process to maintain maximum viability in both strains of mice. The difference in the viability of untreated and nanoinjected embryos is not statistically significant when observing a large number of untreated and nanoinjected zygotes over multiple in vitro experiments. Plotted confidence intervals are Jeffreys 95% confidence intervals for binomial proportions. Supplementary material 3 (EPS 89 kb)

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Copyright information

© Springer Science+Business Media B.V. 2012

Authors and Affiliations

  • Quentin T. Aten
    • 1
  • Brian D. Jensen
    • 1
  • Susan Tamowski
    • 2
  • Aubrey M. Wilson
    • 3
  • Larry L. Howell
    • 1
  • Sandra H. Burnett
    • 3
  1. 1.Mechanical Engineering DepartmentBrigham Young UniversityProvoUSA
  2. 2.Transgenic and Gene Targeting Mouse Core, 2000 Circle of HopeUniversity of UtahSalt Lake CityUSA
  3. 3.Microbiology and Molecular Biology DepartmentBrigham Young UniversityProvoUSA

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