A self-reconfiguring metamorphic nanoinjector for injection into mouse zygotes.

This paper presents a surface-micromachined microelectromechanical system nanoinjector designed to inject DNA into mouse zygotes which are ≈90 µm in diameter. The proposed injection method requires that an electrically charged, DNA coated lance be inserted into the mouse zygote. The nanoinjector's principal design requirements are (1) it must penetrate the lance into the mouse zygote without tearing the cell membranes and (2) maintain electrical connectivity between the lance and a stationary bond pad. These requirements are satisfied through a two-phase, self-reconfiguring metamorphic mechanism. In the first motion subphase a change-point six-bar mechanism elevates the lance to ≈45 µm above the substrate. In the second motion subphase, a compliant folded-beam suspension allows the lance to translate in-plane at a constant height as it penetrates the cell membranes. The viability of embryos following nanoinjection is presented as a metric for quantifying how well the nanoinjector mechanism fulfills its design requirements of penetrating the zygote without causing membrane damage. Viability studies of nearly 3000 nanoinjections resulted in 71.9% of nanoinjected zygotes progressing to the two-cell stage compared to 79.6% of untreated embryos.

Transgene delivery via intracellular electroporetic nanoinjection.

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.

Nanoinjection: pronuclear DNA delivery using a charged lance.

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 µm(2)) is smaller than that of a microinjection pipette tip (0.78 µm(2)). 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.