Gene delivery is the process of introducing foreign DNA into host cells.

Gene delivery is, for

example, one of the steps necessary for gene therapy and the genetic modification of crops.
There are many different methods of gene delivery developed for a various types of cells and
tissues, from bacterial to mammalian. Generally, the methods can be divided into two
categories, viral and non-viral.
Virus mediated gene delivery utilizes ability of a virus to inject its DNA inside a host cell. A
gene that is intended for delivery is packaged into a viral particle.
Non-viral methods include physical methods such as microinjection, gene gun, impalefection,
hydrostatic pressure, electroporation, continuous infusion, and sonication and chemical, such
as lipofection. It can also include the use of polymeric gene carriers.
Microinjection refers to the process of using a very fine needle to insert substances at a
microscopic or borderline macroscopic level into a single living cell. It is a simple
mechanical process in which a needle roughly 0.5 to 5 micrometers in diameter penetrates the
cell membrane and/or the nuclear envelope. The desired contents are then injected into the
desired sub-cellular compartment and the needle is removed. Microinjection is normally
performed under a specialized optical microscope setup called a micromanipulator. The
process is frequently used as a vector in genetic engineering and transgenetics to insert
genetic material into a single cell. Microinjection can also be used in the cloning of
organisms, and in the study of cell biology and viruses.
Examples
• Scientists can create simple transgenic organisms by injecting genes into the testicle
of a nematode at a point where the cells that will become its sperm are undergoing
meiosis. Since the developing gametes share a common cytoplasm, all of the
nematode's gametes will carry a foreign gene as the result of a single injection.
• Microinjection is used as a vector in transgenic plant production.
• Microinjection of genes into fertilized eggs is a common vector used in the production
of higher forms of transgenic animals.
• Microinjection of a gene knockdown reagent such as a Morpholino oligo into eggs or
early zygotes is commonly used to probe the function of a gene during development
of embryos.
• The gene gun or the Biolistic Particle Delivery System, originally designed for plant
transformation, is a device for injecting cells with genetic information . The payload
is an elemental particle of a heavy metal coated with plasmid DNA. This technique is
often simply referred to as biolistics. Another instrument that uses biolistics
technology is the PSD-1000/He particle delivery system (pictured).
• This device is able to transform almost any type of cell, including plants, and is not
limited to genetic material of the nucleus: it can also transform organelles, including
plastids.
Design
The gene gun was originally a Crosman air pistol modified to fire dense tungsten particles.
The design was first used on onions to deliver particles coated with a marker gene. Genetic
transformation can then be proven when the onion tissue expresses the gene. The earliest
custom manufactured geneguns (Fabricated by Nelson Allen) used a 22 caliber nailgun
cartridge to propel an extruded polyethylene cylinder (bullet) down a 22 cal. Douglas barrel.
A droplet of the tungsten powder and genetic material was placed on the bullet and shot down
the barrel at a lexan "stopping" disk with a petri dish below. The bullet welded to the disk and
the genetic information blasted into the sample in the dish with a doughnut effect (devastation
in the middle, a ring of good transformation and little around the edge). The gun was
connected to a vacuum pump and was under vacuum while firing. The early design was put
into limited production by a Rumsey-Loomis (a local machine shop then at Mecklenburg Rd
in Ithaca, NY, USA). Later the design was refined by removing the "surge tank" and changing
to nonexplosive propellants. DuPont added a plastic extrusion to the exterior to visually
improve the machine for mass production to the scientific community. Biorad contracted with
Dupont to manufacture and distribute the device. Improvements include the use of helium
propellant and a multi-disk-collision delivery mechanism. Other heavy metals such as gold
and silver are also used. Gold may be favored because it has better uniformity than tungsten
and tungsten can be toxic to cells, but its use may be limited due to availability and cost.
The primary inventor of the gene gun is horticultural scientist John C. Sanford together with
Edward Wolf, who was the Director of Cornell's Submicron Facility at the time but now at
Nanofabrication facility, and Nelson Allen. As an electrical engineer, Wolf is familiar with
making and using small structures. He bought the Crosman air pistol and performed the first
genegun experiments with it in his basement. Sanford would come to his house with the
genetic material and then take the transformed cells back to his lab. Horticultural scientist
Theodore Klein at Cornell University worked closely with John Sanford on experiments
using and proving the genegun. They had support from co-inventor Nelson Allen of the
Cornell Nanofabrication Facilities Machine shop who had an instrumental role in changing
the genegun from the air pistol prototype to a working scientific device. The rights to
commercial use of the gene gun were sold by Wolf, Sanford and Cornell University to
DuPont in 1990.
Application
Gene guns are so far mostly applied for plants cells. However, there is much potential use in
animals and humans as well.
Plants
The target of a gene gun is often a callus of undifferentiated plant cells growing on gel
medium in a petri dish. After the gold particles have impacted the dish, the gel and callus are
largely disrupted. However, some cells were not obliterated in the impact, and have
successfully enveloped a DNA coated tungsten particle, who’s DNA eventually migrates to
and integrates into a plant chromosome.
Cells from the entire petri dish can be re-collected and selected for successful integration and
expression of new DNA using modern biochemical techniques, such as a using a tandem
selectable gene and northern blots.
Selected single cells from the callus can be treated with a series of plant hormones, such as
auxins and gibberellins, and each may divide and differentiate into the organized, specialized,
tissue cells of an entire plant. This capability of total re-generation is called totipotency. The
new plant that originated from a successfully shot cell may have new genetic (heritable)
traits.
The use of the gene gun may be contrasted with the use of Agrobacterium tumefaciens and its
Ti plasmid to insert genetic information into plant cells. See transformation for different
methods of transformation in different species.
Humans and animals
Gene guns have also been used to deliver DNA vaccines to experimental animals.
Theoretically, it may be used in humans as well.
The delivery of plasmids into rat neurons through the use of a gene gun, specifically DRG
neurons, is also used as a pharmacological precursor in studying the effects of
neurodegenerative diseases such as Alzheimer's Disease.

The Gene gun technique is also popularly used in Edible vaccine production technique, where
the nano gold particles coated with plant gene under the high vacuum pressurized chamber is
transformed into suitable plant tissues.
Electroporation, or electropermeabilization, is a significant increase in the electrical
conductivity and permeability of the cell plasma membrane caused by an externally applied
electrical field. It is usually used in molecular biology as a way of introducing some
substance into a cell, such as loading it with a molecular probe, a drug that can change the
cell's function, or a piece of coding DNA.[1]
Electroporation is a dynamic phenomenon that depends on the local transmembrane voltage
at each cell membrane point. It is generally accepted that for a given pulse duration and
shape, a specific transmembrane voltage threshold exists for the manifestation of the
electroporation phenomenon (from 0.5V to 1V). This leads to the definition of an electric
field magnitude threshold for electroporation (Eth). That is, only the cells within areas where
E≧Eth are electroporated. If a second threshold (Eir) is reached or surpassed, electroporation
will compromise the viability of the cells, i.e., irreversible electroporation.[2]
In molecular biology, the process of electroporation is often used for the transformation of
bacteria, yeast, and plant protoplasts. In addition to the lipid membranes, bacteria also have
cell walls which are different from the lipid membranes and are made of peptidoglycan and
its derivatives. However, the walls are naturally porous and only act as stiff shells that protect
bacteria from severe environmental impacts. If bacteria and plasmids are mixed together, the
plasmids can be transferred into the cell after electroporation. Several hundred volts across a
distance of several millimeters are typically used in this process. Afterwards, the cells have to
be handled carefully until they have had a chance to divide producing new cells that contain
reproduced plasmids. This process is approximately ten times as effective as chemical
transformation.[1][3]
This procedure is also highly efficient for the introduction of foreign genes in tissue culture
cells, especially mammalian cells. For example, it is used in the process of producing
knockout mice, as well as in tumor treatment, gene therapy, and cell-based therapy. The
process of introducing foreign DNAs into eukaryotic cells is known as transfection.

Laboratory Practice
Cuvettes for electroporation. These are plastic with aluminium electrodes and a blue lid. They
hold a maximum of 400 μl.
Electroporation is done with electroporators, appliances which create an electro-magnetic
field in the cell solution. The cell suspension is pipetted into a glass or plastic cuvette which
has two aluminum electrodes on its sides.
For bacterial electroporation, typically a suspension of around 50 microliters is used. Prior to
electroporation it is mixed with the plasmid to be transformed. The mixture is pipetted into
the cuvette, the voltage and capacitance is set and the cuvette inserted into the electroporator.
Immediately after electroporation, one milliliter of liquid medium is added to the bacteria (in
the cuvette or in an eppendorf tube), and the tube is incubated at the bacteria's optimal
temperature for an hour or more to allow recovery of the cells and expression of antibiotic
resistance, followed by spreading on agar plates.
The success of the elecroporation depends greatly on the purity of the plasmid solution,
especially on its salt content. Solutions with high salt concentrations might cause an electrical
discharge (known as arcing), which often reduces the viability of the bacteria.
For a further detailed investigation of the process more attention should be paid to the output
impedance of the porator device and the input impedance of the cells suspension (e.g. salt
content). As the process needs direct electrical contact between the electrodes and the
suspension, and is inoperable with isolated electrodes, obviously the process involves certain
electrolythic effects, due to small currents and not only fields.
The Electroporators
Electroporators come in two flavors - hand-held and bench-tops.
Benchtop electroporators are generally used as common lab equipments, residing atop a
central bench or hood. They offer the advantage of electroporating multiple samples at the
same time. They can also be set to different operating parameters depending on whether the
cell has a cell-wall or not. Unlike them, the handheld electroporators are cordless,
rechargeable and use disposable pipectrodes, which combine elements of both cuvettes and
pipettes. It's operating parameters are pre-set to the optimal parameters for transforming
either bacteria or mammalian cells.
Both types of electoporators have been used on a wide range of cells - including E. coli (for
transformation) and the mammalian cells such as neurons, astrocytes, neuroglia,
lymphocytes, monocytes, fibroblasts, epithelial and endothelial cells from humans, mice, rats
and monkeys (for transfection).
Medical Applications
A higher voltage of electroporation was found in pigs to irreversibly destroy target cells
within a narrow range while leaving neighboring cells unnaffected, and thus represents a
promising new treatment for cancer, heart disease and other disease states that require
removal of tissue
Physical Mechanism
Further information: Lipid bilayer mechanics
Electroporation allows cellular introduction of large highly charged molecules such as DNA
which would never passively diffuse across the hydrophobic bilayer core.[1] This phenomenon
indicates that the mechanism is the creation of nm-scale water-filled holes in the membrane.
Although electroporation and dielectric breakdown both result from application of an electric
field, the mechanisms involved are fundamentally different. In dielectric breakdown the
barrier material is ionized, creating a conductive pathway. The material alteration is thus
chemical in nature. In contrast, during electroporation the lipid molecules are not chemically
altered but simply shift position, opening up a pore which acts as the conductive pathway
through the bilayer as it is filled with water.
Electroporation is a multi-step process with several distinct phases.[5] First, a short electrical
pulse must be applied. Typical parameters would be 300-400mV for <1ms across the
membrane (note- the voltages used in cell experiments are typically much larger because they
are being applied across large distances to the bulk solution so the resulting field across the
actual membrane is only a small fraction of the applied bias). Upon application of this
potential the membrane charges like a capacitor through the migration of ions from the
surrounding solution. Once the critical field is achieved there is a rapid localized
rearrangement in lipid morphology. The resulting structure is believed to be a “pre-pore”
since it is not electrically conductive but leads rapidly to the creation of a conductive pore.
Evidence for the existence of such pre-pores comes mostly from the “flickering” of pores,
which suggests a transition between conductive and insulating states.[6] It has been suggested
that these pre-pores are small (~3Å) hydrophobic defects. If this theory is correct, then the
transition to a conductive state could be explained by a rearrangement at the pore edge, in
which the lipid heads fold over to create a hydrophilic interface. Finally, these conductive
pores can either heal, resealing the bilayer or expand, eventually rupturing it. The resultant
fate depends on whether the critical defect size was exceeded[7] which in turn depends on the
applied field, local mechanical stress and bilayer edge energy.
Lipofection (or liposome transfection) is a technique used to inject genetic material into a
cell by means of liposomes, which are vesicles that can easily merge with the cell membrane
since they are both made of a phospholipid bilayer. Lipofection is a lipid-based transfection
technology which belongs to biochemical methods including also polymers, DEAE dextran
and calcium phosphate. The main advantages of lipofection are its high efficiency, its ability
to transfect all types of nucleic acids in a wide range of cell types, its ease of use,
reproducibility and low toxicity. In addition this method is suitable for all transfection
applications (transient, stable, co-transfection, reverse, sequential or multiple
transfections…), high throughput screening assay and has also shown good efficiency in
some in vivo models.
Sonication is the act of applying sound (usually ultrasound) energy to agitate particles in a
sample, for various purposes. Sonication can be used to speed dissolution, by breaking
intermolecular interactions. In biological applications, sonication may be sufficient to disrupt
or deactivate a biological material. For example, sonication is often used to disrupt cell
membranes and release cellular contents. This process is called sonoporation. Sonication can
also refer to buzz pollination - the process that bees use to shake pollen from flowers by
vibrating their wing muscles.