Download Nanotechnology for Genetic Engineering in Agriculture

ISB NEWS REPORT
JUNE 2013
Nanotechnology for Genetic Engineering in Agriculture
Sandra H. Burnett and Brian D. Jensen
Background
Genetically engineered animals are being produced
annually in ever-expanding numbers for a large variety
of beneficial uses. Genetic engineering can be used to
improve the health, productivity, and quality of food
animals. Agricultural animals can be engineered to
produce biologically active products that might easily
be extracted from milk of a lactating animal for treating
human health conditions. In addition, genetically
engineered animals can be used to research genetic or
infectious diseases where the resultant data provide
insights into animal and human health conditions.
In 1985, pronuclear microinjection was the
first successful method used to produce genetically
engineered animals1. Other methods of producing
genetic modifications now include embryonic stem cell
transfer into blastocysts, somatic cell nuclear transfer,
intracytoplasmic sperm injection (ICSI), and perivitelline
injection of viral-vectors. Even with the range of
methods available for genetic engineering of rodents,
microinjection remains one of the preferred delivery
strategies. Pronuclear microinjection has advantages in
genetic engineering due to early DNA integration that
is ideal for use in precision editing techniques, which
are currently of great interest to engineering goals in
agricultural animals2. Pronuclear microinjection has the
disadvantage of necessitating direct injection of zygotes
that may not be ideal for pronuclear manipulation.
Problems with Pronuclear Microinjection in Agricultural Animals
Pronuclear microinjection requires liquid delivery of
DNA directly into the pronucleus of the fertilized egg.
Pronuclei of mice can typically be identified with good
optics in a microscope, but the view is severely hindered
in agricultural animals due to the cloudiness of zygotes
from sheep, cows, pigs, rabbits, and goats. Researchers
who utilize pronuclear microinjection for genetic
engineering of agricultural animals must cope with the
low visibility of the harvested zygotes. If the DNA is
injected into the cytoplasm and not into the pronucleus,
DNA integration does not occur. Cloudy zygotes can
be centrifuged to shift cytoplasmic contents for better
visualization of pronuclei to improve the ability to inject
into a pronucleus. Centrifugation does not necessarily
clear the view for all zygotes, so only a subset of cells
will be suitable for microinjection after centrifugation.
Centrifugation also has the potential to damage the zygote
and impede proper development.
Solutions in Nanotechnology
Microfabrication was first developed for the manufacturing
of electronic integrated circuits for computer chips. Over
the past two decades, microfabrication has been adapted
and extended to the production of miniature chip systems
referred to as microelectromechanical systems or MEMS.
MEMS can be designed with structures that range from
micrometers to nanometers in size. MEMS can have
moving parts and can also integrate electrical circuits
as part of the features on the chip. Microfabrication has
generated MEMS with a wide variety of applications that
has impacted multiple fields, including sensing, printing,
communications, and biology. In particular, the invention
of techniques to fabricate parts with dimensions on the
same order as the size of cells has stimulated the field of
Bio-MEMS, in which microfabrication is used to explore
and manipulate bio-molecules and cells.
Recently, we developed a new method of delivering
DNA to fertilized mouse eggs using a MEMS chip with
a moveable, nanometer-sized lance that is capable of
holding DNA based on electrical charge3 (Figure 1). We
dubbed the new method “Nanoinjection” based on the
smallness of the lance and the ability to deliver DNA.
Initial studies of nanoinjection demonstrated that the
lance on the MEMS chip could hold an electrical charge
in order to collect DNA on the surface of the lance. The
lance could then be moved forward on the chip using
moveable parts of the MEMS device in order to pierce a
zygote’s pronucleus. The charge on the lance could then
be reversed to release DNA from the tip and surface of
the lance. Withdrawing the lance from the cell would
leave the DNA behind in the pronucleus for integration
into the host cell’s genome (see left panel of Figure 2).
Initial studies demonstrated that pronuclear
nanoinjection had similar DNA integration rates
and significantly higher survival rates compared to
microinjection, giving nanoinjection a measurable
advantage over microinjection for pronuclear delivery
of DNA3. However, pronuclear nanoinjection leaves
the same problem for agricultural animals in that the
pronucleus is still targeted for direct DNA delivery and
unfortunately still requires the ability to visualize a
pronucleus in a cloudy zygote.
The goal of our most recent research with nanoinjection
ISB NEWS REPORT
Figure 1: Electron micrograph of a MEMS nanoinjector. A latex
bead the size of an animal zygote is positioned across from the nanoinjection lance. The lance is able to move across the surface of the MEMS
chip. Electrical connections allow for voltage control of the lance to hold
and release DNA from the lance surface. The scalebar on the bottom left
is 200 um.
Figure 2: Pronuclear nanoinjection includes: A) collection of neg-
atively-charged DNA on the nanoinjection lance with a positive charge, B)
insertion of the lance into the pronucleus and reversal of charge on the
lance to release DNA, C) removal of the lance. Intracellular electroporetic
nanoinjection includes: D) collection of DNA on the nanoinjection lance using charge, E) insertion of the lance into the cytoplasm and reversal of
charge on the lance to generate an electroporation envelope that opens
pores in the pronucleus and electrically repulses DNA away from the lance,
F) removal of the lance.
JUNE 2013
pores can naturally and immediately close upon cessation
of the voltage pulse5. Studies of electroporation by others
have shown that membrane pores open at approximately
200 V/cm, so that a 1 cm cuvette containing cells in
suspension is typically subjected to multiple pulses of
200 Volts. Electroporation has a reputation of causing
high cell death and moderate DNA delivery in tissue
culture cells. Certainly, electroporation has not been
used to generate genetically engineered animals due to
the destructive nature of the method and the fact that
fertilized eggs have a zona pellucida that serves as an
additional protective layer around the cell.
We determined to identify the use of the nanolance
in delivering a localized electrical pulse with an effective
field strength of 200 V/cm. Since the nanoinjection lance
could serve as an electrode, the pathway for current is no
longer a full centimeter, but a drastically smaller distance,
which allows a much lower voltage to deliver a localized
electrical field with a strength of 200 V/cm in a very small
area directly around the lance. Figure 3 shows model
data predicting the envelope around the lance inside
of which the electric field is strong enough to produce
electroporation of pronuclear membranes. We call this
envelope the electroporation envelope. Superimposed on
the data is the cell membrane for a fertilized mouse egg
cell as well as the location and size of the two pronuclei
which we determined using stereo microscopy of 106
mouse embryos4. The figure shows that with a voltage
only 2 V above the decomposition voltage (the voltage
at which electrolysis and electrophoresis begins), the
electroporation envelope is large enough to include at
least part of both pronuclear membranes. Hence, the
model predicts that a very low voltage (less than 10 V)
will be sufficient to open pores in the pronuclei.
was to develop a method of delivering transgene to a
pronucleus without having to physically see or directly
inject the pronucleus. We have accomplished this goal
in our latest method called Intracellular Electroporetic
Nanoinjection (IEN)4.
Principles of Intracellular Electroporetic Nanoinjection
Electroporation is a method used in DNA delivery to
tissue culture cells by subjecting the cells to a strong
electrical field sufficient to disrupt the surface of the cell’s
membranes and open pores for DNA diffusion into the
cell. If the voltage is not too high or too long in duration,
Figure 3: Model data showing the electroporation envelope around
the nanoinjection lance. The data is superimposed over the cell membrane
of a fertilized mouse egg cell. (A) shows an the electroporation envelope in relation to pronuclei within the cell, while (B) shows the electroporation envelope in
addition to the movement of DNA particles from the lance and includes scale
information (in microns).
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Aside from the ability of the lance to generate
localized electroporation, the use of a negative charge
for localized electroporation adds the repulsive forces
of charge to propel negatively-charged DNA that was
collected on the lance surface away from the lance at the
same time as generation of the electroporation envelope.
Computer modeling was again used to predict DNA
repulsion behavior upon reversing the charge on the lance
from a positive to a negative6. The simulations indicated
that 5 ms of repulsion time would repel DNA associated
with the lance a sufficient distance to enter both pronuclei
while at the same time forming the electroporation
envelope around the lance in a manner to open pores for
the discharged DNA to enter the pronuclei.
In
summary,
Intracellular
Electroporetic
Nanoinjection (IEN) is the process of utilizing the
nanoinjection lance to: first, gather DNA on the lance
surface based on charge; second, move the lance into a
central location in the fertilized egg; and third, reverse
the charge in a voltage pulse to generate electroporation
in nearby pronuclei as well as to cast DNA on the lance
away from the lance to enter the pores that temporarily
form in pronuclei (see right panel of Figure 2). The
voltage pulse is short and low so as not to damage the
cell. After pulsing the cell for localized intracellular
electroporation, the lance can be removed. The process of
IEN is done without regard to the location of pronuclei,
so that visual identification or aiming of the pronuclei is
JUNE 2013
not necessary.
We performed a comparative study of IEN and
traditional microinjection in mice. The data indicated
that fertilized eggs and gestation of implanted embryos
experienced no significant difference in viability. We
further found that DNA integration rates were not
different between mice in the IEN group compared to
those in the microinjection group4.
A Promising Future for IEN use in Agricultural
Animals
Our IEN studies demonstrated that DNA was integrated
into mice with no significant difference in integration
rate compared to microinjection4. The fact that IEN
doesn’t offer an integration or survival advantage is far
from discouraging. The fact that IEN works to result in
integration of new DNA is a great advantage because
the pronucleus never needs to be visualized or targeted.
Therefore, IEN holds great promise in application to
genetic engineering in agricultural animals with cloudy
zygotes. Cloudy zygotes would not require centrifugation
if IEN is used to deliver the DNA segments. Application
of IEN has great potential for use in precision editing
techniques for engineering agricultural animals. We look
forward to application of IEN in agricultural animals
to determine the usefulness and validity of this new
nanotechnology for genetic engineering in agriculture.
References
1. Hammer RE, Pursel VG, Rexroad CE Jr, Wall RJ, Bolt DJ, Ebert KM, Palmiter RD, Brinster RL (1985) Production of transgenic rabbits, sheep and pigs by
microinjection. Nature 315(6021):680–683.
2. Tan W, Carlson DF, Walton MW, Fahrenkrug SC, Hacket PB. (2012) Precision Editing of Large Animal Genomes. Adv. Genet. 80:37-97. doi: 10.1016/
B978-0-12-404742-6.00002-8.
3. Aten QT, Jensen BD, Tamowski S, Wilson AM, Howell LL, Burnett SH. (2012) Nanoinjection: pronuclear DNA delivery using a charged lance. Transgenic
Res. 21(6):1279-1290. doi: 10.1007/s11248-012-9610-6
4. Wilson AM, Aten QT, Toone NC, Black JL, Jensen BD, Tamowski S, Howell LL, Burnett ES. (2013) Transgene delivery via intracellular electrophoretic
nanoinjection. Transgenic Res. Epub 20 Mar 2013 doi: 10.1007/s11248-013-9706-7
5. Tsong TY (1991) Electroporation of cell membranes. Biophys J 60(2):297–306. doi:10.1016/s0006-3495(91)82054-9
6. David RA, Jensen BD, Black JL, Burnett SH, Howell LL (2010) Modeling and experimental validation of DNA motion in uniform and nonuniform DC
electric fields. J Nanotechnol Eng Med 1(4):041007-041015. doi:10.1115/1.4002321
Sandra H. Burnett, Ph.D., Associate Professor
Microbiology and Molecular Biology Department
Brian D. Jensen, Ph.D., Associate Professor
Mechanical Engineering Department
Brigham Young University, Provo, UT
[email protected], [email protected]