Gene Delivery Using Physical Methods - Challenges in Delivery of Therapeutic Genomics and Proteomics - page 99

their ratio, dimension and material of carrier particles, method of covering carrier
particles with macromolecules, bombardment force, kind of gene gun, circulation of
carrier particles at target site, kind of targeted cells and tissues, density of carrier par-
ticles and macromolecules. It is widely used in transfection of various biological sys-
tems, like skin and liver in animals, and is also useful to deliver macromolecules in
cancer treatments and AIDS. One of the major applications of the gene gun is genetic
vaccination in various diseases like cancer, influenza virus, bladder pain in animals,
and also used for in vivo and in vitro transfection studies in animals and cell lines.
3.6 Microinjection
Microinjection is a direct method to introduce DNA into either cytoplasm or nucleus.
It is a microsurgical procedure conducted on a single cell, using a glass needle (i.e.,
a fine, glass microcapillary pipette), a precision positioning device (a micromanipu-
lator) to control the movement of the micropipette, and a microinjector. Extrusion
of fluid containing the genetic material through the micropipette uses hydrostatic
pressure. Injections are typically carried out under direct visual control, using a
microscope. The small tip diameters of these micropipettes, combined with the high
precision of the micromanipulator, allow accurate and precise DNA delivery. This
technique is based on the experiments of Barber [198] and forms the basis of devel-
opments observed today [198,199] . Conceptually, microinjection is the simplest gene
delivery method. However, it is difficult to apply. Although pronuclear injection of
DNA is very efficient, it is a laborious procedure; only one cell at a time can be
injected, typically allowing for only a few hundred cells to be transfected per experi-
ment. The cytoplasmic injection of DNA has been observed to be less effective prob-
ably because of cytoplasmic degradation of DNA by cytoplasmic nuclease enzymes.
A more recent development after establishing the microinjection technique is
developing capillary microinjection into cultured somatic cells growing on a solid
support [200] . This technique is now established as one of the most flexible tech-
nique for introducing DNA and even RNA into living cells. These developments
have also helped researchers to study single cells for complicated cellular processes,
structure, and functions in vitro . The microinjection of DNA by capillary injection
into the cells is shown in Figure 3.3 . Microinjection is still widely used to develop
transgenic animals. One of the significant developments in the area of microinjection
gene therapy is automated micromanipulations and microinjection processes as well
as the control and standardization of cell preparation or the production of injection
capillaries [201,202] . Computer-assisted and microprocessor-controlled injection
systems have allowed high injection rates with reproducible results, thus allowing
for quantitative microinjection [203] . Along with nuclear microinjection of DNA, the
microinjection technique has also been utilized for mitochondrial DNA delivery and
cytoplast fusion for gene delivery to treat non-Mendelian genetic diseases caused
by mitochondrial DNA mutations. In this fusion method, mitochondrial DNA in the
cytoplast is transferred into mutant cells through cybrid formation [204] . However,
the injected plasmid DNA is rapidly degraded in the cytoplasm, with an apparent