Agarose gel electrophoresis is a method of choice for the identification, purification, and separation of the DNA fragments. DNA fragments from the gel are routinely extracted for various downstream processing. These include, cloning, radio-labeling, in vitro transcription, microinjection and sequencing of the DNA molecules.
Gel extraction of DNA fragments is mainly done to remove proteins and salts that incorporate from certain reactions. Therefore, in order to use the DNA fragments for downstream processing, these components musts be removed. For example, a PCR amplification or restriction enzyme digestion reaction contains factors which inhibit further applications of the DNA fragment.
There are various methods employed for the extraction of DNA fragments from agarose gel. Among the methods used, silica-membrane containing spin-column based DNA extraction is the most widely used. This is the quickest method to effectively purify DNA fragments from agarose gels.
However, DNA extraction using this method is not always efficient and depends on following factors:
1)Size of the DNA fragment
The first factor affecting the extraction of DNA fragment from the agaorose gel is the size of the DNA fragment itself. It has been observed that DNA fragments of size 500-5000bp are extracted most efficiently from the agarose gels. However, DNA fragments smaller than 500bp or lager than 5000bp, are poorly recovered from the gel.
2)Concentration of agarose in the gel
Concentration of agarose in the gel has a significant effect on the extraction of DNA fragments. It has been observed that recovery of DNA fragments is inversely proportional to the concentration of agarose in the gel. As the concentration of agarose in the gel increases, recovery of DNA molecules decreases. Therefore, in order to carryout good recovery of DNA fragments, less concentration of agarose is used in the gel.
Generally, for gel extraction, 0.7-1% agarose gels are prepared. However, if the size of the DNA fragment is less than 300bp you will have to use higher concentration of agarose (1.2-1.5%) in the gel. We routinely use 0.7% agarose gels for extracting DNA molecules.
3)Voltage applied to the gel
The applied voltage also significantly affects the extraction of DNA fragments. In order to extract any DNA molecule from the gel you need to excise it out. For this you need a sharp and distinct band of specific DNA in the gel. Therefore, to get a sharp band, the gel has to be run using the appropriate voltage.
It has been observed that 3-5V/cm is the optimum voltage for DNA extraction. Increasing or decreasing the voltage will result in poor resolution and hence the poor recovery of the DNA fragments. For extracting the DNA fragments, we generally apply a voltage of 3V/cm to the gel.
4)Cutting of the gel slice
Cutting the gel slice also has a significant effect on the recovery of the DNA molecules. You should cut the gel slice just adjacent to the band. Moreover, extra gel portions if any should be carefully removed. It has been found that extra gel portions greatly reduce the recovery of DNA molecules.
In addition, you should try to cut small gel slices for extraction purpose. Lager gel slices incompletely solubilize and hence results in the poor recovery of the DNA molecules.
5)pH of the extraction buffer
As the adsorption of DNA fragments to silica membranes largely depends on pH, therefore, extraction of DNA fragments is significantly affected by the pH of the extraction buffer. It has been observed that maximum adsorption occurs at pH less than 7.5. As the pH of the buffer increases, adsorption of DNA to silica membrane is reduced drastically.
In order to monitor the pH of the extraction buffer, many commercially available kits add indicators to the extraction buffer. If the pH increases, color of the buffer changes, thereby enabling easy monitoring of the pH.
There are certain reasons for the increase of pH of the extraction buffer. For example, agarose gel is having incorrectly prepared high pH buffer, or the electrophoresis buffer had been repeatedly used, etc. In these cases, the pH of the extraction buffer can easily be corrected by adding a small volume of 3 M sodium acetate, pH 5.2, before proceeding.
6)Residual ethanol
After the adsorption of the DNA molecules to the silica membrane, the membrane is washed with solutions having 70% ethanol. However, before the addition of elution buffer to the membrane, the residual ethanol must be removed. It has been observed that presence of residual ethanol on the silica membrane greatly reduces the recovery of the DNA fragments.
In order to remove the residual ethanol the best way is to centrifuge the column for two minutes at maximum rpm. However, we have observed that doing so doesn’t completely remove the residual ethanol. Therefore, you should keep the column at room temperature for a while to remove the residual ethanol. But be careful, as over-drying the membrane will, in turn, result in lower recovery of the DNA fragments.
7)pH and salt concentration of the elution buffer
Elution efficiency of the DNA fragments from silica membranes strongly depends on the salt concentration and pH of the elution buffer. It has been found that most efficient elution is carried out under alkaline pH (7.5-8.5) and low salt concentrations.
Generally, the DNA fragments are eluted using 10 mM Tris-Cl, pH 8.5. However, TE buffer is not recommended for the elution of the DNA because some downstream processing may get inhibited by the presence of EDTA. Apart from 10 mM Tris-Cl, pH 8.5, MQ grade water can also be used to elute the DNA fragments. However, you will have to make sure that the pH of the water remains in the range of 7.5-8.5. Moreover, recovery of DNA fragments will be less if water is used instead of the elution buffer.
8)Volume of elution buffer
Generally, elution of DNA fragments in a spin column based extraction is carried out in small volumes. However, the volume of the elution buffer plays a significant role in the recovery of the DNA molecules. It has been observed that most efficient elution is carried out using 20-50µl of the elution buffer.
Using elution buffer less than 20µl will result in poor recovery of the DNA fragments. However, using lager volumes will result in the elution of less concentrated DNA fragments. For various purposes, we generally elute the DNA fragments in 25µl of elution buffer.
9)Incubation time of the buffer on the column
Since the main purpose of the elution buffer is to provide a medium in which the DNA fragments will be dissolved. Therefore, incubating the spin column after the addition of the elution buffer has a significant role in the recovery of the DNA molecules. Generally, it is a good practice to incubate the column for 2-5 minutes prior to centrifugation.
You can clearly see that there are a number of factors influencing the extraction of DNA fragments from agarose gel using spin column based method. Each of the above listed factors should be properly considered in order to achieve efficient extraction of the DNA.
Recently, the first "zoofarming" product has reached market
approval: it is a recombinant human protein for medical use that is
produced in the milk of transgenic goats. In addition, other transgenic
animals, including faster-growing salmon and „environmentally
friendly" pigs with reduced levels of phosphate in their feces are
awaiting regulatory approval. These are only some examples of upcoming
applications of genetic engineering in farm animals. Other potential
applications include traditional breeding goals such as higher milk or
meat yields, leaner meat, and disease resistance. While genetic
engineering in livestock opens a huge range of possibilities, it also
brings about concerns of safety and justification: does genetic
engineering affect animal welfare? Is it safe and morally acceptable to
apply genetic engineering to farm animals for the various purposes
that are envisaged? It is against this background that
the Europäische Akademie GmbH and the Berlin-Brandenburgische
Akademie der Wissenschaften addressed the topic of transgenic farm
animals in an interdisciplinary symposium in 2007. In these
proceedings the following topics are covered: an analysis of the state
of the art of the technology and its applications, an introduction to
the specific application zoopharming (including its historical
industrial development and the market for biopharmaceuticals), an
assessment of ethical aspects, and considerations regarding the
investigation of animal welfare implications of livestock
biotechnology. The proceedings address science, industry, politics and
the general public interested in the chances and risks of this
upcoming field of biotechnology.
Angiogenesis is a normal process in growth and development, as well as
in wound healing. However, this is also a fundamental step in the
transition of tumors from a dormant state to a malignant state.
Sprouting angiogenesis was the first
identified form of angiogenesis. It occurs in several well-characterized
stages. First, biological signals known as angiogenic growth factors
activate receptors present on endothelial cells present in pre-existing
venular blood vessels. Second, the activated endothelial cells begin to
release enzymes called proteases that degrade the basement membrane in
order to allow endothelial cells to escape from the original (parent)
vessel walls. The endothelial cells then proliferate into the
surrounding matrix and form solid sprouts connecting neighboring
vessels. As sprouts extend toward the source of the angiogenic stimulus,
endothelial cells migrate in tandem, using adhesion molecules, the
equivalent of cellular grappling hooks, called integrins. These sprouts
then form loops to become a full-fledged vessel lumen as cells migrate
to the site of angiogenesis. Sprouting occurs at a rate of several
millimeters per day, and enables new vessels to grow across gaps in the
vasculature. It is markedly different from splitting angiogenesis,
however, because it forms entirely new vessels as opposed to splitting
existing vessels.
Intussusception, also known as
splitting angiogenesis, was first observed in neonatal rats. In this
type of vessel formation, the capillary wall extends into the lumen to
split a single vessel in two. There are four phases of intussusceptive
angiogenesis. First, the two opposing capillary walls establish a zone
of contact. Second, the endothelial cell junctions are reorganized and
the vessel bilayer is perforated to allow growth factors and cells to
penetrate into the lumen. Third, a core is formed between the two new
vessels at the zone of contact that is filled with pericytes and
myofibroblasts. These cells begin laying collagen fibers into the core
to provide an extracellular matrix for growth of the vessel lumen.
Finally, the core is fleshed out with no alterations to the basic
structure. Intussusception is important because it is a reorganization
of existing cells. It allows a vast increase in the number of
capillaries without a corresponding increase in the number of
endothelial cells. This is especially important in embryonic development
as there are not enough resources to create a rich microvasculature
with new cells every time a new vessel develops.
Modern Terminology of Angiogenesis
Besides the differentiation between
“Sprouting angiogenesis” and “Intussusceptive angiogenesis” there exists
the today more common differentiation between the following types of
angiogenesis:
Vasculogenesis
– Formation of vascular structures from circulating or tissue-resident
endothelial stem cells (angioblasts), which proliferate into de-novo
endothelial cells. This form particularly relates to the embryonal
development of the vascular system.
Angiogenesis –
Formation of thin-walled endothelium-lined structures with/without
muscular smooth muscle wall and pericytes (fibrocytes). This form plays
an important role during the adult life span, also as "repair mechanism"
of damaged tissues.
Arteriogenesis – Formation of medium-sized blood vessels possessing tunica media plus adventitia.
Because it turned out that even this
differentiation is not a sharp one, today quite often the term
“Angiogenesis” is used summarizing all different types and modifications
of arterial vessel growth.
Therapeutic angiogenesis
Therapeutic angiogenesis is the
application of specific compounds which may inhibit or induce the
creation of new blood vessels in the body in order to combat disease.
The presence of blood vessels where there should be none may affect the
mechanical properties of a tissue, increasing the likelihood of failure.
The absence of blood vessels in a repairing or otherwise metabolically
active tissue may retard repair or some other function. Several diseases
(eg. ischemic chronic wounds) are the result of failure or insufficient
blood vessel formation and may be treated by a local expansion of blood
vessels, thus bringing new nutrients to the site, facilitating repair.
Other diseases, such as age-related macular degeneration, may be created
by a local expansion of blood vessels, interfering with normal
physiological processes.
The modern clinical application of the
principle “angiogenesis” can be divided into two main areas: 1.
Anti-angiogenic therapies (historically, research started with); 2.
Pro-angiogenic therapies. Whereas anti-angiogenic therapies are trying
to fight cancer and malignancies(because tumors, in general, are
nutrition- and oxygen-dependent, thus being in need of adequate blood
supply), the pro-angiogenic therapies are becoming more and more
important in the search of new treatment options for cardiovascular
diseases (the number one cause of death in the Western world). One of
the world-wide first applications of usage of pro-angiogenic methods in
humans was a German trial using fibroblast growth factor 1 (FGF-1) for
the treatment of coronary artery disease. Today, clinical research is
ongoing in various clinical trials to promote therapeutic angiogenesis
for a variety of atherosclerotic diseases, like coronary heart disease,
peripheral arterial disease, wound healing disorders, etc.
Also, regarding the “mode of action”,
pro-angiogenic methods can be differentiated into three main categories:
1. Gene-therapy; 2. Protein-therapy (using angiogenic growth factors
like FGF-1 or vascular endothelial growth factor, VEGF); 3. Cell-based
therapies.
There are still serious, unsolved
problems related to gene therapy including: 1. Difficulty integrating
the therapeutic DNA (gene) into the genome of target cells; 2. Risk of
an undesired immune response; 3 Potential toxicity, immunogenicity,
inflammatory responses and oncogenesis related to the viral vectors; and
4. The most commonly occurring disorders in humans such as heart
disease, high blood pressure, diabetes, Alzheimer’s disease are most
likely caused by the combined effects of variations in many genes, and
thus injecting a single gene will not be beneficial in these diseases.
In contrast, pro-angiogenic protein therapy uses well defined, precisely
structured proteins, with previously defined optimal doses of the
individual protein for disease states, and with well-known biological
effects. On the other hand, an obstacle of protein therapy is the mode
of delivery: oral, intravenous, intra-arterial, or intramuscular routes
of the protein’s administration are not always as effective as desired;
the therapeutic protein can be metabolized or cleared before it can
enter the target tissue. Cell-based pro-angiogenic therapies are still
in an early stage of research – with many open questions regarding best
cell types and dosages to use.
FGF
The fibroblast growth factor (FGF)
family with its prototype members FGF-1 (acidic FGF) and FGF-2 (basic
FGF) consists to date of at least 22 known members. Most are 16-18 kDa
single chain peptides and display high affinity to heparin and heparan
sulfate. In general, FGFs stimulate a variety of cellular functions by
binding to cell surface FGF-receptors in the presence of heparin
proteoglycans. The FGF-receptor family is comprised of seven members and
all the receptor proteins are single chain receptor tyrosine kinases
that become activated through autophosphorylation induced by a mechanism
of FGF mediated receptor dimerization. Receptor activation gives rise
to a signal transduction cascade that leads to gene activation and
diverse biological responses, including cell differentiation,
proliferation, and matrix dissolution – thus initiating a process of
mitogenic activity critical for the growth of endothelial cells,
fibroblasts, and smooth muscle cells. FGF-1, unique among all 22 members
of the FGF family, can bind to all seven FGF-receptor subtypes, making
it the broadest acting member of the FGF family, and a potent mitogen
for the diverse cell types needed to mount an angiogenic response in
damaged (hypoxic) tissues, where up regulation of FGF-receptors occurs.
FGF-1 stimulates the proliferation and differentiation of all cell types
necessary for building an arterial vessel, including endothelial cells
and smooth muscle cells; this fact distinguishes FGF-1 from other
pro-angiogenic growth factors, such as vascular endothelial growth
factor (VEGF) which primarily drives the formation of new capillaries.
Until now (2007), three human clinical
trials have been successfully completed with FGF-1 in which the
angiogenic protein was injected directly into the damaged heart muscle.
Also, one additional human FGF-1 trial has been completed to promote
wound healing in diabetics with chronic wounds.
Besides FGF-1, one of the most
important functions of also fibroblast growth factor-2 (FGF-2 or bFGF)
is the promotion of endothelial cell proliferation and the physical
organization of endothelial cells into tube-like structures, thus
promoting angiogenesis. FGF-2 is a more potent angiogenic factor than
VEGF or PDGF (platelet-derived growth factor), however, less potent than
FGF-1. As well as stimulating blood vessel growth, aFGF (FGF-1) and
bFGF (FGF-2) are important players in wound healing. They stimulate the
proliferation of fibroblasts and endothelial cells that give rise to
angiogenesis and developing granulation tissue, both increase blood
supply and fill up a wound space/cavity early in the wound healing
process.
VEGF
VEGF (Vascular Endothelial Growth
Factor) has been demonstrated to be a major contributor to angiogenesis,
increasing the number of capillaries in a given network. Initial in
vitro studies demonstrated that bovine capillary endothelial cells will
proliferate and show signs of tube structures upon stimulation by VEGF
and bFGF, although the results were more pronounced with VEGF.
Upregulation of VEGF is a major component of the physiological response
to exercise and its role in angiogenesis is suspected to be a possible
treatment in vascular injuries. In vitro studies clearly demonstrate
that VEGF is a potent stimulator of angiogenesis because in the presence
of this growth factor plated endothelial cells will proliferate and
migrate, eventually forming tube structures resembling capillaries. VEGF
causes a massive signaling cascade in endothelial cells. Binding to
VEGF receptor-2 (VEGFR-2) starts a tyrosine kinase signaling cascade
that stimulates the production of factors that variously stimulate
vessel permeability (eNOS, producting NO), proliferation/survival
(bFGF), migration (ICAMs/VCAMs/MMPs) and finally differentiation into
mature blood vessels. Mechanically, VEGF is upregulated with muscle
contractions as a result of increased blood flow to affected areas. The
increased flow also causes a large increase in the mRNA production of
VEGF receptors 1 and 2. The increase in receptor production means that
muscle contractions could cause upregulation of the signaling cascade
relating to angiogenesis. As part of the angiogenic signaling cascade,
NO is widely considered to be a major contributor to the angiogenic
response because inhibition of NO significantly reduces the effects of
angiogenic growth factors. However, inhibition of NO during exercise
does not inhibit angiogenesis indicating that there are other factors
involved in the angiogenic response.
Angiopoietins
The angiopoietins, Ang1 and Ang2, are
required for the formation of mature blood vessels, as demonstrated by
mouse knock out studies . Ang1 and Ang2 are protein growth factors which
act by binding their receptors, Tie-1 and Tie-2; while this is somewhat
controversial, it seems that cell signals are transmitted mostly by
Tie-2; though some papers show physiologic signaling via Tie-1 as well.
These receptors are tyrosine kinases. Thus, they can initiate cell
signaling when ligand binding causes a dimerization that initiates
phosphorylation on key tyrosines.
MMP
Another major contributor to
angiogenesis is matrix metalloproteinase (MMP). MMPs help degrade the
proteins that keep the vessel walls solid. This proteolysis allows the
endothelial cells to escape into the interstitial matrix as seen in
sprouting angiogenesis. Inhibition of MMPs prevents the formation of new
capillaries. These enzymes are highly regulated during the vessel
formation process because destruction of the extracellular matrix would
decrease the integrity of the microvasculature.
Applications of Angiogenesis Tumor angiogenesis
Cancer cells are cells that have lost
their ability to divide in a controlled fashion. A tumor consists of a
population of rapidly dividing and growing cancer cells. Mutations
rapidly accrue within the population. These mutations (variation) allow
the cancer cells (or sub-populations of cancer cells within a tumor) to
develop drug resistance and escape therapy. Tumors cannot grow beyond a
certain size, generally 1-2 mm³, due to a lack of oxygen and other
essential nutrients.
Tumors induce blood vessel growth
(angiogenesis) by secreting various growth factors (e.g. Vascular
Endothelial Growth Factor or VEGF). Growth factors, such as bFGF and
VEGF can induce capillary growth into the tumor, which some researchers
suspect supply required nutrients -- allowing for tumor expansion. On 18
July 2007 it was discovered that cancerous cells stop producing the
anti-VEGF enzyme PKG. In normal cells (but not in cancerous ones), PKG
apparently limits beta-catenin which solicits angiogenesis.Other
clinicians believe that angiogenesis really serves as a waste pathway,
taking away the biological end products put out by rapidly dividing
cancer cells. In either case, angiogenesis is a necessary and required
step for transition from a small harmless cluster of cells, often said
to be about the size of the metal ball at the end of a ball-point pen,
to a large tumor. Angiogenesis is also required for the spread of a
tumor, or metastasis. Single cancer cells can break away from an
established solid tumor, enter the blood vessel, and be carried to a
distant site, where they can implant and begin the growth of a secondary
tumor. Evidence now suggests that the blood vessel in a given solid
tumor may in fact be mosaic vessels, comprised of endothelial cells and
tumor cells. This mosaicity allows for substantial shedding of tumor
cells into the vasculature. The subsequent growth of such metastases
will also require a supply of nutrients and oxygen or a waste disposal
pathway.
Endothelial cells have long been
considered genetically more stable than cancer cells. This genomic
stability confers an advantage to targeting endothelial cells using
antiangiogenic therapy, compared to chemotherapy directed at cancer
cells, which rapidly mutate and acquire 'drug resistance' to treatment.
For this reason, endothelial cells are thought to be an ideal target for
therapies directed against them. Recent studies by Klagsbrun, et al.
have shown, however, that endothelial cells growing within tumors do
carry genetic abnormalities. Thus, tumor vessels have the theoretical
potential for developing acquired resistance to drugs. This is a new
area of angiogenesis research being actively pursued.
Angiogenesis research is a cutting
edge field in cancer research, and recent evidence also suggests that
traditional therapies, such as radiation therapy, may actually work in
part by targeting the genomically stable endothelial cell compartment,
rather than the genomically unstable tumor cell compartment. New blood
vessel formation is a relatively fragile process, subject to disruptive
interference at several levels. In short, the therapy is the selection
agent which is being used to kill a cell compartment. Tumor cells evolve
resistance rapidly due to rapid generation time (days) and genomic
instability (variation), whereas endothelial cells are a good target
because of a long generation time (months) and genomic stability (low
variation).
This is an example of selection in
action at the cellular level, using a selection pressure to target and
differentiate between varying populations of cells. The end result is
the extinction of one species or population of cells (endothelial
cells), followed by the collapse of the ecosystem (the tumor) due either
to nutrient deprivation or self-pollution from the destruction of
necessary waste pathways.
Angiogenesis-based tumour therapy
relies on natural and synthetic angiogenesis inhibitors like
angiostatin, endostatin and tumstatin. These are proteins that mainly
originate as specific fragments pre-existing structural proteins like
collagen or plasminogen.
Recently, the 1st FDA-approved therapy
targeted at angiogenesis in cancer came on the market in the US. This
is a monoclonal antibody directed against an isoform of VEGF. The
commercial name of this antibody is Avastin, and the therapy has been
approved for use in colorectal cancer in combination with established
chemotherapy.
Angiogenesis for cardiovascular disease
Angiogenesis represents an excellent
therapeutic target for the treatment of cardiovascular disease. It is a
potent, physiological process that underlies the natural manner in which
our bodies respond to a diminution of blood supply to vital organs,
namely the production of new collateral vessels to overcome the ischemic
insult. Perhaps the greatest reason for these trials’ failure to
achieve success is the high occurrence of the “placebo effect” in
studies employing treadmill exercise test readout. Thus, even though a
majority of the treated patients in these trials experience relief of
such clinical symptoms such as chest pain (angina), and generally
performed better on most efficacy readouts, there were enough
“responders” in the blinded placebo groups to render the trial
inconclusive. In addition to the placebo effect, more recent animal
studies have also highlighted various factors that may inhibit an
angiogenesis response including certain drugs, smoking, and
hypercholesterolemia.
Although shown to be relatively safe
therapies, not one angiogenic therapeutic has yet made it through the
gauntlet of clinical testing required for drug approval. By capitalizing
on the large database of what did and did not work in previous clinical
trials, results from more recent studies with redesigned clinical
protocols give renewed hope that angiogenesis therapy will be a
treatment choice for sufferers of cardiovascular disease resulting from
occluded and/or stenotic vessels.
Early clinical studies with
protein-based therapeutics largely focused on the intravenous or
intracoronary administration of a particular growth factor to stimulate
angiogenesis in the affected tissue or organ. Most of these trials did
not achieve statistically significant improvements in their clinical
endpoints. This ultimately led to an abandonment of this approach and a
widespread belief in the field that protein therapy, especially with a
single agent, was not a viable option to treat ischemic cardiovascular
disease. However, the failure of gene- or cell-based therapy to deliver,
as of yet, a suitable treatment choice for diseases resulting from poor
blood flow, has led to a resurgence of interest in returning to
protein-based therapy to stimulate angiogenesis. Lessons learned from
earlier protein-based studies, which indicated that intravenous or
intracoronary delivery of the protein was not efficacious, have led to
completed and ongoing clinical trials in which the angiogenic protein is
injected directly into the beating ischemic heart.
Such localized administration of the
potent angiogenic growth factor, human FGF-1, has recently given
promising results in clinical trials in no-option heart patients.
Angiogenesis was documented by angiographically visible “blushing”, and
functional exercise tests were also performed on a subset of patients.
The attractiveness of protein therapy is that large amounts of the
therapeutic agent can be injected into the ischemic area of interest, to
pharmacologically start the process of blood vessel growth and
collateral arteries’ formation. In addition, from pharmacokinetic data
collected from the recent FGF-1 studies in the human heart, it appears
that FGF-1, once it exits the heart is cleared in less than three hours
from the circulation. This would presumably prevent FGF-1 from
stimulating unwanted angiogenesis in other tissues of the bodies where
it could potentially cause harm, such as the retina and in the kidneys.
No serious adverse events have yet to be noted in any of the completed
or ongoing clinical trials in which the FGF-1 protein is utilized as the
therapeutic agent tom stimulate angiogenesis
