Articles on this Page
- 10/12/12--06:22: _A Guide for Best Ed...
- 10/13/12--03:48: _Cloning: Simple exp...
- 10/13/12--04:06: _Plants Bioreactors ...
- 10/13/12--10:16: _Cloning of endanger...
- 10/14/12--05:35: _Transgenic animals:...
- 10/14/12--10:34: _Genetically modifie...
- 10/15/12--04:30: _Overview of Gene Th...
- 10/15/12--05:41: _Vesicular Trafficki...
- 10/15/12--10:23: _Eradicate Malaria U...
- 10/16/12--03:29: _Can cells be grown ...
- 10/16/12--23:24: _Preservatives - A P...
- 10/19/12--21:56: _Microbes for Fighti...
- 10/21/12--23:15: _Test Methods to Stu...
- 10/22/12--02:36: _Applications of Gen...
- 10/22/12--10:07: _Transgenic Plants /...
- 10/23/12--00:13: _Role of Type 2 Rest...
- 10/23/12--05:55: _Antisense RNA Techn...
- 10/23/12--23:06: _A Study on Culture ...
- 10/25/12--01:05: _DNA Microarrays: DN...
- 10/25/12--02:18: _Role of Environment...
- 10/12/12--06:22: A Guide for Best Education in Biotech: Popular Colleges in USA
- 10/13/12--03:48: Cloning: Simple explanation through examples
- 10/13/12--04:06: Plants Bioreactors and Their Classification
- 10/13/12--10:16: Cloning of endangered and extinct species
- 10/14/12--05:35: Transgenic animals: how they are created and what’s their purpose
- 10/14/12--10:34: Genetically modified plants and their application
- 10/15/12--04:30: Overview of Gene Therapy and its implications in medical field
- 10/15/12--05:41: Vesicular Trafficking - A Major Pathway of Neurotransmission
- 10/15/12--10:23: Eradicate Malaria Using Genetic Engineering
- 10/16/12--23:24: Preservatives - A Possible Solution for Biodeterioration
- 10/19/12--21:56: Microbes for Fighting Tooth Decay? What do Researchers Say?
- 10/21/12--23:15: Test Methods to Study Genotoxic Compounds
- 10/22/12--02:36: Applications of Genetic Engineering in Biotechnology
- 10/22/12--10:07: Transgenic Plants / Genetically Modified Crops | In-Depth Analysis
- 10/23/12--00:13: Role of Type 2 Restriction Endonucleases in Gene Cloning
- 10/23/12--05:55: Antisense RNA Technology and its Applications
- 10/23/12--23:06: A Study on Culture and Production of Artificial Skin
- 10/25/12--01:05: DNA Microarrays: DNA on a Chip
- 10/25/12--02:18: Role of Environmental factors in Gene Expression
If you search for best biotechnology colleges in USA on the internet, you'll get couple of lists with completely different names mentioned. I prefer list created by user rating. Hope you'll find given info useful.
University is located in West Lafayette, Indiana. It’s established in 1868, thanks to generous donation of land and money by local businessman, John Purdue. It all started with 6 professors and 39 students, and today – Purdue is second largest college in Indiana. In 2010, 6,614 academic staff and 39,726 students were enrolled in various study programs. With 210 major areas of study, students can earn degree in different scientific, technological and agricultural disciplines.
Leland Stanford Junior University (full name) is located in Stanford, California. It’s founded in 1831 by Governor of California, in honor of his son who died of typhoid. Until 1930 tuition was free of charge. It survived San Francisco earthquake and several money crises. Today, that is prestigious college with over 40 Nobel Prizes won. Google, Yahoo, Hewlett Packard … are just few examples of the companies founded by the faculty members. University is divided in 7 schools: humanities and sciences, earth sciences, schools of business, education, engineering, law and medicine.
University of California San Diego
UCSD is located in La Jolla, California, near the Pacific Ocean. More than 23000 undergraduates and 5500 graduates are enrolled in ~200 study programs. It’s one of the best public undergraduate colleges (Public Ivy) in America. University operates 4 research institutes. For the past three years, it’s ranked No 1 University in nation by Washington Monthly. Successful research in oceanography, molecular biology and genetics, neuroscience and behavior, global warming phenomena, as well as Keeling Curve and green fluorescent protein discoveries are just few things that UCSD is famous for.
BU is located in Boston, Massachusetts. That is private university with over 4000 teachers and 31000 students enrolled in study programs in 18 schools divided in two campuses. Martin Luther King earned PhD at BU and one out of the 7 Nobel Prizes that BU won. University is truly “green”, focused on proper waste management and recycling, energy efficiency and sustainable building development. BU is famous for its high research activity, just in 2009-2010 research expenditures were > 407.8 million dollars.
University of California Davis
UCD is located in Davis, California. It’s public research university, established in 1905. Nobel and Pulitzer Prizes, National Medal of Science, Presidential Early Career Award in Science and Engineering…are just some of the honors won by university members over the years. UCD undergraduate programs can be divided in 4 major categories (colleges): Agricultural and Environmental Sciences, Biological Science, Engineering and Letters & Science. UCD is listed as Public Ivy. Thanks to sustainability and climate change efforts, Sierra Magazine ranked UCD No 1 “coolest” in 2012 and “Newsweek” ranked it 10th “happiest” and 11th “greenest” school in USA in 2011.
Oregon State University
OSU is located in Corvallis, Oregon. Students (> 23000) can choose between 200 undergraduate programs. Microbiology, ecology, forestry, biochemistry, zoology, oceanography, food science and pharmacy…are some of the most popular. Jurassic Park story (and movie) is inspired by the OSU’s entomology professor George Poinar, Jr. and his work on DNA extraction from insects fossilized in amber.
Rensselaer Polytechnic Institute
RPI is located in Troy, New York. It’s private research university founded in 1824. University is divided in 5 schools that are offering 140 degree programs in 60 different fields. RPI is ranked No 7 by salary potential after graduation and it’s among top 50 national universities. Research centers operated by RPI are focused on biotechnology, energy & environment and nanotechnology, among other technical fields. James Fallon, adult stem cell pioneer, earned his diploma at RPI.
Columbia University is located in New York, New York. It’s founded as King’s college in 1754 and it’s considered to be one of the private Ivy colleges. Columbia University encompasses 20 schools and is lying on more than 6 city blocks. Some students became very rich and successful (20 living billionaires), powerful (29 heads of state, including 3 presidents) or famous (25 Academy Award winners) after graduating from Columbia. ~30 currently marketed medical products, green fluorescent protein labeling, 600 patents and 250 active license agreements…are showing how creative and fruitful work on Columbia can be.
Each of this school will provide necessary amount of knowledge, you just need to pick one that is oriented the most toward your favorite field of science and biotechnology.
When you think of cloning, Dolly the sheep is probably the first thing that comes to your mind. Artificial cloning came later; we stole that idea from nature. Asexual reproduction, typical for so many animals, is natural way to reproduce. New individual is created by division of the mother cell giving daughter cell with the same genetic material. Biotechnology found a way to utilize this natural process for production of novel molecules, cells or even organisms. Main purpose is to help solve certain medical issues or reveal genetic mysteries in various experiments that are taking place all over the world.
Molecular cloning is used to amplify DNA sequence (gene, promoter, non-coding sequence…) of interest. To ensure replication, DNA sequence must be linked with origin of replication – part of DNA that will initiate replication. Ligation is process of inserting DNA sequence into cloning vector (peace of DNA, carrier of the sequence). DNA ligaze will connect sequence and vector by “gluing” sticky ends at each DNA piece. Transfection of cell with vector carrying sequence of interest is next step. Electroporation, optical injection or biolistics are mostly used transfection techniques, but they are not successful always. Additional genes in cloning vector are necessary to ensure easy recognition of cells containing DNA piece of interest. Some of the most famous “markers” used are genes providing antibiotic resistance (when substrate with antibiotic is used for cell growing) or color markers (for blue/white cell screening). After cell colonies are formed, DNA sequence will be multiplied and analyzed using PCR, DNA sequencing or restriction fragment analysis.
Cellular cloning is production of cells containing same genetic info as mother cell. Cells derived from multi-cellular organism are much more complicated to clone than cells that are unicellular by nature. Technique “clone rings” is used for cloning multi-cellular organism derived cells. Cell suspension is exposed to mutagenic agent or drug and planted at high dilution, which result in new colonies formation. On a stage of few cells created, trypsin and polystyrene rings (covered in grease) are placed over each formed colony. Cells from the inner part of the ring are collected and moved to another substrate to develop further. Cellular cloning could solve serious medical issues that are non-treatable by conventional medication (such as Alzheimer disease). Cells used for this purpose are stem cells - as they could give raise to any cell lineage we want. SCNT (Stem Cell Nuclear Transport) is used for developing embryonic stem cells (ESC) that will have both research and therapeutic application. ESC are created by removing nucleus from the egg and implanting nucleus from adult somatic cell (containing both mother and father genetic material). Egg will act like it’s been fertilized and start dividing first to reach blastocyst stage and then toward any cell lineage we want. Procedure is the same with animal species and could be used to produce additional food source (by cloning farm animals) or to prevent extinction of endangered animals. It may sound like simple process, but success rate with this kind of genetic manipulation is pretty low. Dolly the sheep was first mammal created in laboratory. Out of 277 eggs used for SCNT, just 29 embryos were created. 3 survived until birth and only one - more famous by its given name – Dolly, survived until adulthood. Although, genetic material in the newly formed cell (organism) is the same as in donor’s cell, certain part of DNA is unique. Each cell contains mitochondria with its own genetic material. It’s inherited solely from the mother due to couple of reasons: egg contains more mtDNA than sperm; sperm derived mtDNA is easily degraded once inside or it can even fail to enter the egg. Thanks to this phenomenon, cloned cells can be considered genetic hybrids, as they contain both somatic DNA and mother mitochondrial genes.
Some large animals can create clones on their own. Lizards, snakes, ants, crustaceous species and even certain sharks are able to produce new individual by parthenogenesis – out of unfertilized egg. For most species, this is not obligatory way to reproduce but a method to overcome crisis in their environment. Komodo dragon, for example, can reproduce by parthenogenesis to increase the population in the habitat and then switch back to sexual reproduction to increase genetic diversity of the next generation.
To conclude, cloning is not something we invented, it’s natural phenomenon that we start exploiting recently. Wise and careful approach could be beneficial for the planet; we just need to pay attention not to cross the line, as genetic diversity is what allowed us to survive so far.
A Bioreactor is a device or vessel in which are designed to provide an effective environment for conversion of one material into some product by appropriate biochemical reactions and this conversion is carried out by the action of a biocatalysts like enzymes, microorganisms, cells of animals and plants, or subcellular structures such as chloroplasts and mitochondria. There are different bioreactors and they have different applications are including those for cell growth, enzyme production, biocatalysis, food production, milk processing, tissue engineering, algae production, protein synthesis, and anaerobic digestion. Bioreactors are classified depending on their operational conditions and the nature of the process. Bioreactors can be from different sources like animals, plants, microbes,etc.
Plants as bioreactors-
Plant cells are an attractive heterologous expression host for foreign protein production. These are unique biocatalysts that have characters different from microorganisms or animal cells. Plants have become economically important systems for producing heterologous proteins. Expressing heterologous proteins in plant material that is used in human food or animal feed allows proteins to be applied orally or topically without having to purify them from the plant material. Plants have a distinct advantage for these applications. Thus, the recombinant products have an advantage over traditional microbial or mammalian host systems and the other features of plant cells as a production host along with are the cost-effective biomanufacturing and the capacity for complex protein post-translational modifications. Heterologous proteins like therapeutics, antibodies, vaccines and enzymes are expressed in plant cell culture-based bioreactor systems including suspended dedifferentiated plant cells, moss, and hairy roots, etc. The in vitro liquid cultures of plant cells in a fully contained bioreactor have become very promising alternative to traditional microbial fermentation and mammalian cell cultures as a foreign protein expression platform. These plant bioreactors are mainly used to produce therapeutic proteins, edible vaccines and antibodies for immunotherapy.
There are two basic processes that are used to produce recombinant proteins in plants one is generating the transgenic plants by stable integration of transgene into plant genome and the other is transient expression of the transgene using plant viruses as vectors. The other techniques used for direct gene transfer are electroporation, polythene glycol mediated gene uptake and particle bombardment.
There are different plant bioreactors classified based on where the protein is produced-
Plant suspension cultures-
In this plant cells are grown under sterile conditions as suspension or callus cultures and given the appropriate hormonal supplements for growth and are used in expression of recombinant proteins, secondary metabolites and antibodies.
The nuclear chromosomes of chloroplasts are inserted with the foreign genes that are responsible for required product.
Insulin, interferons and other proteins can be prepared in chloroplast bioreactor.
Hairy root system bioreactor-
This has rhizosecretion caused due to infection of agro bacterium rhizogenes and is highly stable and suitable for different biopharmaceuticals.
Seed based plant bioreactors-
Seed is the most suitable bioreactor because of their large protein accumulation during its development. But specificity of expression and subcellular storage environment are the factors that will decide which seeds are used for producing desired products.
There are two types of seed based plant bioreactors-
Seed protein storage vacuole bioreactors-
The protein storage vacuoles in seeds contain some dominant sub compartments like matrix, globoid and crystalloids which are best for storing recombinant protein. Matrix is suitable for soluble storage proteins, globoids for hydrolytic enzymes and crystalloids for some intrinsic protein sequences.
Seed oil body bioreactors-
This bioreactor can store a large amount of macromolecules. It has oleosin proteins which are ideal carriers of heterologous proteins encircling the seed oil body. This also provides recognition signal for lipase binding during oil mobilization in seedlings.
Achieving the highest possible level of foreign protein production is transgenic plant is very important and this needs to have a strong promoter sequence that can enhance the expression of interest. These plant bioreactors with their unique features show not only advantages but also some disadvantages.
They are cost effective, faster than transgenic animals, can produce large biomass and the pathogens do not effect animals and humans.
The difference in codons of prokaryotes and plants can lead to inefficient expression, different polysaccharides may be attached to proteins and some plants may contain allergic compounds.
Technology is developing rapidly in the last decade and so are genetic tools and methods used for genome sequencing, creating transgenic organisms, cloning...Reproductive cloning is one of the most criticized and controversial technique used for creating genetically identical animals. Clone is exact genetic replica of organism that donated the cell (nucleus) used for cloning. A lot of animals are cloned so far. Dolly was first mammal that was made in laboratory, but cat, dog, goat, mule…followed soon after. Success in creating living animal clones made scientist think about cloning extinct animals. Although not one extinct animal is resurrected by now, we are closer that we think to see them in the near future.
In 1972, Oliver Ryder, geneticist at San Diego Zoo decided to collect as much animal skin samples as possible, hoping that tissue bank might be useful in future attempts to save endangered animals. He didn’t know that animals will be created using somatic cells in the future. Stored tissues were named “Frozen Zoo” thanks to preservation method used (liquid nitrogen). Until now, this collection grew to impressive number of ~9 000 samples belonging to ~1 000 different vertebrate species. Although initial idea wasn’t to create a gene pool that will be used for animal cloning, latest techniques and high rate of extinction made “Frozen Zoo” serving that purpose exactly.
First attempts to clone threatened animal species started at the beginning of the 2001 using South Asian ox – guar. SCNT (stem cell nuclear transfer) using guar’s somatic cells and cow’s egg resulted in successful embryo development, that was seeded into cow’s uterus. Noah was born seemingly healthy, but 2 days later – he died due to infection.
In 2000, Spanish ibex, known as bucardo, went extinct. That was the first time that scientist tried to clone extinct animal. Nucleus derived from ibex’s skin cell was implanted into de-nucleated egg of domestic goat - its closest relative. New ibex was born, but died soon after the birth due to severe lung defect. As with Dolly the sheep, a lot of attempts were made before viable embryo that could survive until the birth was created. In the case of bucardo, 439 SCNT were made, 57 embryos were implanted, 7 embryos resulted in pregnancy and just one managed to survive until the birth. He lived for 7 minutes: inability to breath normally prevent him to live longer.
Success rate of cloning is 1%. Beside the low success rate, born animals are unhealthy and prone to various infections that doesn’t keep them alive for a long period.
In 2007, Japanese scientist concluded experiments on mice, revealing that adult somatic cells could be reverted to embryo like stem cells. Those cells were named “induced pluripotent stem cells” (iPS cells) and they could be used for creating any cell lineage you want. Oliver Ryder and his co-workers from San Diego Zoo are using iPS cells to create Northern white rhino, snow leopard and small West African monkey replicas as the number of those species is incredibly low (7 remaining individuals of white rhino are kept in captivity).
Besides “saving” animals that are still alive, or that are extinct few years ago, scientific appetites are growing bigger. How about creating Woolly Mammoth using DNA from his leftovers found under Siberian permafrost recently? Japanese and Russian scientist promise to create a mammoth in couple of years, using mammoth’s nucleus and elephant’s egg. Only problem with resurrecting mammoth is age of the DNA and damage found in his genetic material. However, using modern methods, 80% of mammoth genome is decoded. Creating chimera animal could solve the problem: stem cell derived from mammoth (using iPS method) placed near elephant embryo would affect early embryo development, resulting in animal having tissues created out of both mammoth and elephant cells. We are closer that we think to see mammoth (probably weak and unhealthy, but still successfully created) walking the Earth again.
What is making me really angry and sad is that mankind is responsible for all recently (couple of hundred years) noted extinctions. Why don't we use money and effort made in cloning animals to prevent extinction instead? Don’t you think that those kinds of experiments are cruel to the animals? Who ask female elephant if she is willing to be a surrogate mother to a mammoth? Would you like to be a surrogate mother to a chimpanzee, as its closest relative? I don’t think so. We managed to destroy so many beautiful things on the planet Earth, but driving animals extinct instead preserving them and then resurrecting them just to show future generation how mammoth looked like before he went extinct - I just don’t get it! If human ever become critically endangered – please don't clone me!
Transgenic or genetically modified animals are created when foreign DNA sequence is incorporated in their genome. Main purpose is to provide more information on human and/or animal related diseases, to enhance novel therapeutics development, to assess environmental pollution….
Whatever the purpose, first part of the experiment is always the same: DNA piece need to be inserted into animal’s genome. Mouse is often used for all kind of genetic experiments. Transgenic mouse can be created using two methods: transforming embryonic stem cell or by injecting DNA sequence into pronucleus of fertilized egg.
Mouse blastocyst derived stem cells are transfected with DNA sequence of interest. Sequence is attached to a vector, promoter and enhancer, to ensure proper functioning of the gene in the host’s genome. Successfully transfected embryonic stem cells are injected back into mouse blastocyst. Before implanting embryo in mouse, female is mating with sterile male to ensure hormonal changes necessary for uterus to accept embryo (mating is a trigger). 1 out of 3 implanted embryos survive until birth. After they are born, tissue sample will show if animal is carrying a gene of interest. Less than 20% of offspring will be positive for the gene tested and they will be heterozygous for that gene (present in only one copy of the gene). Mating two heterozygous animals will result in 25% percent of homozygous offspring. They have two copies of desired gene (both from mother and father) and that is the moment when new, transgenic, animal is created. Gene of interest starts to express on a regular basis.
Second method uses sperm head for incorporating sequence of interest. Prepared DNA piece (with vector, promoter…) is injected into male pronucleus before he fuses with egg’s pronucleus. At the stage of 2 cells, embryo will be implanted into female’s uterus (prepared the same way like in stem cell method) for further development.
These methods are applicable to a lot of animals and can be beneficial in medicine (to heal different protein deficiencies). For example, Alpha1-Antitrypsin Deficiency is genetic disorder resulting in lung damage. Transgenic goats carrying alpha1-antitrypsin gene were successfully created but high expenses of protein extraction and purification from goat’s milk prevented company to produce it on a large scale. One other company managed to overcome these difficulties and as from 2006 human antithrombin expressed in goat’s milk is widely used to prevent blood clotting during surgery.
Severe combined immunodeficiency disorder (SCID), known as bubble boy disease, is genetic disorder resulting in inability of organism to fight even slight infections. A lot of babies die in the first year of life due to severe and recurring infections. So far, bone marrow transplantation is only solution and it needs to be provided in the first few month of baby’s life of even in utero (before baby is born). Other solution that proved to be effective (at least at the beginning) was gene therapy using viral vectors “equipped” with gene of interest. Hematopoietic stem cells “enriched” with missing gene helped 4-year old girl to cope with SCID. Later studies showed that this kind of therapy could induce leukemia as retrovirus carrying a sequence of interest could trigger expression of the nearby oncogene as well. Today, efforts are made to produce viral vector that will not affect oncogenesis. A list of disorders that could be treated with gene therapy is long (muscular dystrophy, Parkinson disease, cystic fibrosis…) and even thought solutions are not perfect yet - this medical field is constantly improving.
Besides using transgenic animals or cells for therapeutic purposes, they could serve as model organisms for studying various disorders: triggering genes responsible for disease, mechanism of illness could be observed. Knock out animals are carrying dysfunctional genes that are not expressing proteins. They are designed to detect role and value of different proteins, enzymes, hormones… in the body. When protein expression is lacking, affected biochemical process and/or subsequent disorder will be easily recognized.
Some pets are hypo-allergic - genetically modified to prevent allergic reaction. Farm animals could be “improved” to grow faster or to digest some food that normally wouldn’t be able to do. Green fluorescent protein (GFP) can be perfect marker of environmental pollution once incorporated in zebrafish genome. It is also massively used as a marker of genetic expression, for analysis of neuronal activity, as a viability assay (in cryobiology)... Insects can be genetically altered as well: development of mosquitoes resistant to malaria and incorporation of lethal genes in male mosquitoes responsible for Dengue fever help combat these issues in affected areas.
When it comes to transgenic animals, my emotions are mixed: I encourage getting new knowledge and providing solutions that could help planet as a whole to become a better place, but idea that so many animals are sacrificed for that purpose is making me so sad. I hope that we'll find out that more benefits than damage is made, when we summarize everything we've done.
Ever since the beginning of the plant breeding, man was altering plants to ensure high production and best quality of the harvest. At the beginning, selective and cross-breeding were methods used, but for the last couple of decades man is using more sophisticated methods while designing the plants with all characteristics needed. Genetic engineering became popular in no time and genetically modified plants start sprouting all over that planet.
First experiments were focused on development of the plants resistant to pesticides, to prolong ripening time (tomato), to improve oil composition (canola) and by the 1996 - 8 transgenic plants were approved for cultivation. At the beginning of the 2000, one plant was modified for the first time to increase its nutritional value (golden rise).
Methods used for modification of the plant genome are relatively simple: either Agrobacterium tumefaciens or biolistic gun could be used. In the first case, A. tumefaciens will act as a vector, carrying and incorporating genes of interest in the plant’s genome. This method is useful for dicotyledonous plants like tomato and tobacco. Biolistic method uses DNA sequence attached to the gold or tungsten particles that are shoot into plant cell or tissue using high pressure. After penetrate the cell, DNA is released from the metal particle and start incorporates into the genome. Main disadvantage of this method is mechanical damage inflicted to the cells. However, this method is useful for plants that can’t be easily transfected with A. tumefaciens, like wheat or maize.
Plants could carry genes that will enable them to produce antigens for vaccinations, bacterial toxins or enzyme that will be used for therapeutic purposes. Carrot producing Taliglucerase alfa is used for Gaucher’s Disease treatment; banana producing vaccine against Hepatitis B virus is developed but not marketed; tobacco deriving therapeutic antibodies are under investigation…
Droughts, low temperatures, lack of nutrients are stressful environmental conditions that could negatively affect plant development. Plant could be modified to increase tolerance and survive drastic weather changes.
Herbicides are strong chemicals used to eliminate weed, allowing plant of interest to grow smoothly. Weed could become resistant to an herbicide over time, and genetic engineering is used to create a plant carrying more than one herbicide resistance gene that will allow regular spraying of the crop with multiple herbicides.
Insects and viruses could produce serious damage on the plants. Bacillus thuringiensis derived genes are incorporated into plant’s genome to ensure resistance against insects. Papaya production was dramatically reduced when ringspot virus starts spreading. Genetic modification solved the problem, and it’s still only solution against ringspot virus.
Golden rice is first genetically modified plant with increased nutritional value. Over 650 000 children under the age of 5 are dying each year due to vitamin A deficiency. Designing the rise enriched in beta-carotene gene with resulting high provitamin A level offered solution to the most affected areas. It’s not marketed yet.
Genetic engineering of the plants is useful way of producing biofuels. Algae are well known source of biomass that could be used for the biofuel production. Genetically modified maize that is accelerating ethanol production by converting its own starch into sugar is next promising candidate for biofuel production.
Pollution of the planet is one of the most important issues that mankind is facing today. Every solution that could help preserve environment or help reduce amount of existing waste is more than welcome. Genetically modified crop could be used in bioplastic development. Transgenic plants carrying bacterial genes are cleaning up environment from pollutants like mercury, selenium, PCBs…using bacterial enzymes that are digesting contaminants from the soil.
Genetically modified crops are hundred billion dollars worth business. A lot of people and steps are involved in crop development: from scientist that are experimenting with the plants, companies that are producing the seed, chemical industries developing herbicide and pesticide all the way to the farmers that are cultivating the plant…In 2010, 15 million farmers in 29 countries worldwide were growing genetically modified crops. Over 80% of cultivated corn, cotton and soya in the USA are genetically modified. Beside USA, Brasil, Argentina and India are the largest manufacturers of the genetically modified crops.
What plant will be modified, cultivated, used as a food source…depend on the country and the laws associated with genetically modified organisms. Europe has more strict legislation than USA for example, but still it produces GM crop as well. A lot of people are concerned how this food will affect their health and “health” of surrounding ecosystem. I’m concerned as well, but unfortunately most of us can’t cultivate organic plants and stay healthy for a longer period of time. I know that this is not way too comforting, but what doesn't kill you makes you stronger. To make things worse - I'm vegetarian
The progress in the field of Recombinant DNA technology (RDT) has initiated the discovery of new medicines and therapeutics for various diseases. It has also helped in the diagnosis and detection of different infections, genetic diseases and Cancer. The nucleic acid-based biopharmaceuticals, which includes gene therapy and antisense therapy, have great potential to create a revolution in the field of medical science. However, their role in the medical field can materialise completely only after solving the different difficulties encountered in their successful implication.
Gene therapy can be utilized for the correction of different genetic diseases resulting due to the presence of a defective gene, which arises due to mutation or is inborn. The therapy works on the principle of the introduction of the stable gene, to correct the faulty gene expression or provide protective function, into the genetic complement of the cell with the use of different vectors useful for the purpose. The detailed understanding about the different molecular mechanisms related to the diseases within the body helps in the successful application of the gene therapy in combating the diseases. Due to the adaptation of the human body through evolution to resist the entry of foreign genetic material within its genome has posed a problem for the use of gene therapy. However, the use of viruses as vectors for the introduction of genes into the body has helped in overcoming the problem largely due to the ability of the viruses to overcome the barriers and incorporate their genetic material into the genome of the human cells.
There are three categories of somatic gene therapy:
i) ex-vivo, in which the cells removed from the body are incubated with vectors and then the modified, genetically engineered cells are re-introduced into the body. However, this procedure is restricted with blood cells, which can be removed from and re-introduced into the body.
ii) In-situ, in which the introduction of the vector carrying the gene is done into the affected tissues. This procedure is useful in the treatment of muscular dystrophy, which involves the introduction of the vector carrying the gene of dystrophin into the muscle and the treatment of cystic fibrosis, involving the introduction of the vector expressing the gene of cytokine or toxin into the tumor-infected muscle.
iii) In-vivo, in which the vectors carrying genes are introduced directly into the blood stream of the infected individual. This process has not been applied clinically yet, however to apply gene therapy for therapeutics, the development of in-vivo introduction of injectable vectors is essential.
For gene therapy, two types of vectors have been studied: RNA virus vectors and DNA virus vectors. The Retroviruses were initially chosen as the best vehicle for gene transfer due to a number of advantages they provided such as efficient gene transfer with stable integration into the host cell genome, providing the possibility of long-term expression. As they have evolved into non-pathogenic parasites, hence have minimal risk in their usage. These vectors are replication-defective due to the removal of all their viral genes. However, there are some problems faced by these vectors such as for obtaining proper efficient delivery into the target cells, transduction of the non-dividing cells, sustainment of long-term gene expression, and the development of a cost-effective method of manufacturing the viral vector. DNA viruses, which have been most often used for gene transfer are Adeno virus, Adeno associated virus (AAV), herpes simplex virus, etc. The immune response of the cells against the viruses, which affect the integration into the host genome as also the non-specific integration of the gene are some of the problems faced by the DNA virus vectors. Hence, to solve the problem of generation of immune response, non-viral vectors like liposomes and polypeptides are being developed, although their low transduction efficiency poses the biggest disadvantage of their use.
Thus, it can be said that although gene therapy has potential to be developed as therapeutics for a number of genetic diseases, however the application has some major limitations, due to which it has not found practical application, like:
i)the complexity of the genetic diseases affecting a number of cells and tissues at the same time and the molecular mechanisms involved with the development of the disease,
ii)the insufficiency in the level of gene expression and its regulation,
iii)improper identification of the actual genes responsible for a particular disease, and
iv)the insufficiency of the patient population to be studied for various diseases and for conducting clinical trials.
Hence, although gene therapy is making a very slow progress in being used as a form of treatment, it shows great promise and in future, the modification in the vectors used and the development of better strategies for the purpose will help in the practical application of gene therapy in therapeutics.
The cellular membrane structure is a stable protein-lipid bilayer; however, it is not static in nature. The transport and the movement of the secretory proteins as well as different factors occur continuously across the membrane. The unique feature of the membranes to fuse with other membranes without losing continuity and the continuous reorganization of the membranes within the eukaryotic endo-membrane system is responsible for many of the biological functions of the membranes like transport, secretion, etc.
The specific membrane fusion between two membranes requires:
i) The recognition between the two membranes
ii) Apposition of the membranes with the removal of water molecules associated with the lipid polar head group
iii) The disruption in the bilayer structures resulting in hemifusion, i.e. fusion of the outer leaflet of each membrane
iv) The formation of continuous bilayer with the fusion of the two bilayers.
The triggering of the fusion between the bilayers at the appropriate time or in response to a particular and specific signal is required for the process of receptor-mediated endocytosis or for regulated secretion of various factors or proteins. These events are mediated by integral proteins in the membranes or within the cells called fusion proteins, which bring about specific recognition and the formation of a local distortion transiently for favouring membrane fusion. The secretion of neurotransmitters into the synapse and the process of neurotransmission is one of the most widely studied cases of membrane fusion.
The lipids play a very essential role in the process of exocytosis, being the building blocks of the membrane structure, as the distortion in the bilayer arrangement of the lipid in the membranes helps in the merging of the membranes during the process of fusion. It is facilitated by the presence of non-cylindrical lipids like lysolipids, phosphatidic acid, fatty acids, etc. The cellular concentration of these lipids is low in resting stage and increases with the stimulation and secretion of different lipases. The fact that lysolipids play an important role in exocytosis is supported by their presence in high concentration in the vesicles of the neuroendocrine cells and in the stimulated exocytosis of the neurotransmitters with the Phospholipase A2 secretion. The machinery involving the zippering of the N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) proteins has proved to be capable of mediating the membrane fusion in-vitro. Hence, the role of proteins is also very essential in the membrane fusion process.
The electric impulses travel across the axon of the neurons and the signals are transmitted to the next neuron or the target effectors or cells in the form of the neurotransmitters. These neurotransmitters are contained in the vesicles, which are present within the neurons. A Synapse is the junction between two neurons or between a neuron and its target cell and helps in the transmission of the signals through the synaptic cleft. In the resting stage, it is believed that the synaptic vesicles are loaded with neurotransmitters and they either remain free in the cytoplasm linked to the actin filaments with the help of synapsin bridges or remain docked in the pre-synaptic membrane. The main mechanism of the transmission occurs due to the depolarization of the pre-synaptic membrane leading to the opening of the voltage-gated Ca2+ channels, which triggers the entry of Ca2+ into the pre-synaptic membrane. This results in the fusion of the synaptic vesicles with the pre-synaptic membrane, thereby resulting in the release of the neurotransmitters into the synaptic cleft. These neurotransmitters then bind to the receptors on the post-synaptic membrane resulting in the opening of the ion-gated channels, which leads to the entry of Na+, K+, Ca2+, Cl- into the post-synaptic membrane, depending upon the selectivity of the ion channel. After the fusion and release of the neurotransmitters, the vesicle membranes are retrieved rapidly and are reutilized for the generation of further neurotransmitter-loaded vesicles. The ions, which enter through the opening of the voltage-gated channels or ion-gated channels are used for further cell signalling mechanism. This machinery of the exo-endocytic recycling coordination requires complex mechanisms integrating cell signal transduction, other dynamic changes within the membrane structure and rearrangements of the cytoskeletal elements within the cell.
Although, much research has been conducted regarding the study of the synaptic transmission through the vesicular traffic process, all the proteins involved in the exocytosis and endocytosis of the synaptic vesicles are not known yet. Recent evidence has suggested the role of Synaptotagmin, a phopholipid dependent Ca2+ binding protein involved in the exocytosis of the vesicles. However, the possible involvement of other proteins in the process is yet to be discovered. Proper understanding of the molecular mechanism of the fusion of the membranes of synaptic vesicles and pre-synaptic membrane can be achieved only through the identification and characterization of the different components of the fusion complex formed during the interaction of the synaptic vesicle with the pre-synaptic membrane.
Mosquito, now you may bite me. Thank you genetic engineering!
It has been estimated that 3 million people die of malaria disease. Majority of them are children’s under five year age. Accordingly malaria kills 3,000 children’s per day. This number is significantly more than AIDS. As we all known that malaria is caused by four species of Plasmodium protozoa parasites namely Plasmodium vivax, P. falciparum, P. ovale and P.malaria. The most dangerous is P. falciparum. The mosquito does not cause the malaria but is a vehicle for transfer of these parasites which actually cause disease. The Anopheline mosquitoes are responsible for transfer of these parasites.
In spite of combination therapies, including artesunate and mefloquine drugs etc, the complete eradication of malaria is yet not possible. But the new developments in biotechnology field have shown a promising technology which cans completely eradicate malaria. Biotechnology is employing the concept & knowledge of life cycle of parasite with respect to Anopheline mosquitoes. It has been known by research that the parasite need more than 2 weeks to be able to reproduce and completely establish in mosquito while the age of mosquito is only 3 weeks. Therefore, this is a clue, biotechnology has been successful in genetically modifying few of these Anopheline mosquitoes which reduced their life span by few days, such that the parasites cannot reproduce and establish themselves in mosquitoes as they need complete two weeks for the same and mosquitoes die before it happens. Thus even though mosquito’s bites, they will not transfer viable parasites as required for infection and establishment purpose. Neither the mosquitoes will be infected nor the humans. Once again thank you GE and biotechnology, for tomorrow you may save more than 3000 children’s a day from malaria!
i am new to this site. so dont know if it is the relevant thread to post my question. Can cells be grown in exogenous supply of Malonyl-CoA and Malate. These are the first product/precursor of the first product of two major pathways and i want to check the effect of inhibition of these two pathways. please someone throw some light here. help would be highly appreciated.
Bio-deterioration is process that involves undesirable change in the natural or other economically important material due to the activities of living beings, plants, animals or microorganisms. The process of bio-deterioration is associated with the negative role of microorganisms in the economically useful products. To inhibit the growth of the microorganisms in the useful products, preservatives in different forms are added to the product. A substance, which may be naturally occurring or synthetically produced, added to different products such as food, medicines, pharmaceutical products, paints, wood, different biological samples, etc to prevent them from biodegradation or deterioration or any other undesirable change by the microorganisms, is known as a preservative. They generally inhibit the growth of the microorganisms or kill them in rare cases.
Antimicrobial preservatives are chemical agents that are normally static in nature i.e. they inhibit the growth of the microorganisms in food, pharmaceuticals or cosmetic products that may be ingested. In most of the countries, based on the approval issued by FDA (Food and Drug Association), there are three categories of preservatives. In Group 1, the natural organic acids like lactic acid, citric acid, etc are included, while in Group 2, the substances, which have been classified as generally regarded as safe (GRAS), such as a) other organic acids such as benzoic acid, sorbic acid, etc., b) the parabens such as esters of para-hydroxybenzoic acid, and c) SO2 are included. The compounds, which do not belong to either Group 1 or Group 2 are included in Group 3. There are some compounds such as common salt, spices, vinegar, oil, etc, which act like preservative, but cannot be classified as ‘added preservatives’, due to the difference in their properties in comparison to the added preservatives. The main properties of the added preservatives in food are that they should be safe, and should be free from any sort of flavour or aroma. They should be added in very low concentration and must not be used to disguise the food from its poor manufacturing practice.
The action of the preservatives depends on the pH of the product, to which they are added. The undissociated form of the preservatives possesses anti-microbial activity. As the pH increases, it is seen that the anti-microbial activity of the compounds reduces, except sorbic acid and parabens, which possess anti-microbial activity until pH 6.5 and pH7.0-8.0 respectively. Hence, parabens have found wide application as preservative in food, pharmaceuticals, and cosmetics due to their favourable properties such as being colourless, tasteless, and stable, apart from their wide pH range. They also have high lethal dose as studied on rats. Lethal dose or LD50 can be defined as the dose of the substance in grams per kilograms of body weight of test animals at which 50% of the population are killed. The apparent drawbacks of using parabens are the low-grade sensitization as exhibited by some individuals ingesting it, their increased tendency to exhibit partition into oil, particularly vegetable oils and also their inability to show complete efficacy in presence of ethylene glycol, glycerol, etc. Although, the parabens inhibit the growth of many types of bacteria, yeasts, fungi, etc., they have been found to be almost ineffective against Gram-negative bacteria. The main action of the parabens is assumed to be on the microbial membranes, however they also affect the nucleic acid metabolism. They are used in mixtures for their synergistic action.
Apart from the organic acids, very few microbial products have found application as preservatives. The antibiotics are generally not used as preservatives due to the development of possible resistance and also the probable disturbance of the microbial ecology in the gut, toxicity, or different allergic reactions. The development of the antibiotics for use as preservatives is a lengthy, expensive and complex procedure. The long-term safety and toxicological study are also major concerns in their development. Natamycin, an antifungal agent that acts as surface antimycotic agent for cut and sliced foods, e.g. cheese and Nisin, a bacteriocin that is effective in heat processed foods and low pH foods, are the only antibiotic agents to be approved for use as food preservatives. Nisin is effective on the gram-positive bacteria but is very less effective against the gram-negative bacteria and yeast. Other biological preservatives used widely are lactoferrin; avidin, a biotin-chelator; ovoinhibitor, an inhibitor of protease; and lactoperoxidase, an SH group oxidiser. Lysozyme has also been used as a preservative, though it has limited bacteriolytic activity spectrum, for bacterial cell lysis. Chitin, a cell wall component of fungi has also found application as a food preservative.
Apart from food, pharmaceuticals and cosmetics, and other economically useful products such as wood, etc have also been found prone to biodeterioration. Due to biodeterioration, the active ingredient in the pharmaceuticals and cosmetics is lost making it useless and in some cases even toxic for use. Hence, the use of non-toxic preservatives in minute concentration have been recommended such that the products remain sterile for a longer period of time and are also safe for use. However, in-depth study about the conditions necessary, the nature of preservatives and its effect on the formulation of the products is very essential.
Bacteria living on teeth convert sugar into lactic acid which erodes enamel & causes decay. Florida-based company on biopharma has engineered a new bacterial strain called smart that can’t produce lactic acid. But it releases ankills the natural decay causing strain.
In a bid to fight tooth decay, researchers have come up with new weapon in the form of a bacterium that produces an enzyme which inhibits the formation of plaque.
There are as many as 500 different species of bacteria that inhabit our mouth and can colonize on your teeth and gums. When we have a meal, these bacteria forms layers called biotims on the teeth which helps to convert sugars sugars like sucrose, fructose and glucose.left on your teeth and gums to acids. This process leads to production of lactic acid which breaks down tooth enamel and leads to cavities. Streptococcus mutans and Lactobacillus are found to be the most cariogenic (promotes tooth decay) of these bacteria.
There are other beneficial bacteria like Streptococcus salivarius that is found on the tongue and soft tissues of the mouth and it is known to fight the biofilms built up by Streptococcus mutans.
A researcher in this study, accomplished the task of turning cavity causing bacteria into cavity fighting bacteria by stripping the bacterium of its ability to produce lactic acid. It is this byproduct of the breakdown of sugar by Streptococcus mutans that causes tooth decay. If the bacteria are not able to produce lactic acid tooth decay is stopped.
The genetically altered strain of Streptococcus mutans appeared to thrive on sugar. Researchers found that the strain was able to stay on the surface of the teeth indefinitely and prevented the natural strain from colonizing on the teeth. The altered strain is genetically stable and no ill effects have been noted.
Dentists will only need to swab smart now in clinical trials onto tooth once to keep them healthy for a lifetime. The bacteria convert the glucose, fructose & sucrose into lactic acid through a glycolytic process.
Other researchers in Tokyo discovered that an off-the-shelf FruA acquired from fungus Aspergillus niger also has the similar qualities despite the fact that it has a different amino acid sequence to the one found in the mouth. They used chromatography technique to isolate proteins from S. salivarius and these are used in to find out what was responsible for its cavity fighting powers. Cultures were prepared and mixed with S. mutans cells. The culture with protein FruA had the smallest biofilm and this was evidence that it was the most powerful biofilm blocker.
According to a report in Applied and Environmental Microbiology the researchers found that when there is increased sucrose concentration in the mixtures containing S. salivarius FruA and S. mutans their ability to prevent biofilm formation is decreased.
Another research in UK suggests that using microbes to fight against microbes or more precisely an enzyme from bacteria found on the surface of seaweed. Lab tests have shown that the enzyme is effective in fighting plaque.
An enzyme isolated from Bacillus licheniformis was identified during a screening for compounds that could disperse microbes from the surfaces of ship hulls. When under threat, these bacteria create a slimy protective biofilm barrier of extracellular DNA that joins them together while also sticking to a solid surface. This sticky matrix offers the microbes some protection from brushing, chemical washes or even antibiotics.
These researchers discovered that the enzyme could break down the external DNA, weakening and breaking up the biofilm layer so that the bacteria could no longer find a foot-hold and so get evicted. Initial experiments in the lab have shown promise in demonstrating that the enzyme has the ability to cut through plaque but more tests are scheduled to prove the discovery is both effective and safe.
The components of these discoveries may be used as ingredients in a paste, mouthwash or denture cleaning product. Although it could take a few years before anything appears on the shelves of local pharmacies the scientists say that these could also be useful for keeping certain medical implants clean. But researchers warn that this does not mean that you can get rid of your tooth brush. Brushing and other forms of dental hygiene would still be recommended to prevent plaque build-up.
Genotoxicity, a branch of toxicology is developed to identify the elements or compounds present in the environment having the potential to cause mutation by damaging the DNA. These compounds are also classified under the group of carcinogens, because of their cancer causing property. The necessity to identify the toxic compounds causing mutation is important in various industries like pharmaceutical, agriculture and food as the end user of the products from these industries are humans. As a result various methods were developed to detect and assess the toxic elements. The conventional method of using laboratory animals like mice and rat as test subjects is replaced by newly developed in vitro methods using microorganisms (bacteria) and animal cells. Few of such mostly used testing methods include Ames test, cell line tests and cytogenetic or chromosomal test.
Ames Test: Ames test employs bacteria in detecting the mutagenecity of the test compound. The mostly used species is mutant of Salmonella typhimurium, the ability to produce histidine of this organism is altered by mutating histidine operon gene present in this bacterium. The mutant organism is plated on an agar plate prepared with small amount of added histidine and the test element is placed at the centre of the plate using a filter disc. The toxicity of the compound is assessed by the growth of the bacteria. Initially, the bacterium grows till the presence of added histidine. Later, only those organisms whose mutation is reversed by the test compound, grows by producing histidine. This is qualitative test. The amount of compound required to cause mutation is quantified with the help of dose-response curve.
Later two types of mutations like single base substitution and frame shift mutation were adopted to create mutant organisms with the scope of studying more number of suspected toxic compounds. The sensitivity of the test is enhanced by altering the permeable nature of the bacteria and their cell repair mechanism. The use of bacteria as the test organism to study the mutagenic elements or compounds that are threat to humans has its own limitations. For example, once the compound enters the body, its toxicity is established only after the action of certain enzymes produced in the human body. To overcome this problem, extracts of liver with active enzymes were added along with the test element.
Use of Cell lines: The fact that the testing of Genotoxicity of an element on mammals is more beneficial rather than the use of bacteria, led to the use of mammalian cell lines in vitro. The cell lines derived from the mouse lymphocyte is used and the thymidine kinase heterozygote test is considered as the popular method of toxicity testing. The mouse cell lines are selected based on the mutation occurred on the thymidine kinase gene locus. The mutants are derived by treating the cells with toxic copies of thymidine. The selected cells are grown in the cell culture media by exposing them to the compound under study.
Chromosome test or cytogenetic test: This method involves the use of cell lines. The mutagenic property of the test compound is detected by observing the cells for chromosomal damage. Earlier, the mutagenic property of the test element is assessed by calculating the chromosomal damages like chromosomal breakages, chromosomal exchange, formation of ring chromosome, chromosomal dicentric and chromosomal translocation. Later, due to the difficulty in assessing the toxicity by this method, enumeration of sister chromatid exchange is done to detect the element for its mutagenic property. The increase in the frequency of sister chromatid exchange on exposing to mutagens excited the researchers and they preferred this method than counting the chromosomal aberrations. The sister chromatid are stained to study the type of exchange taken place using the fluorescence Giemsa staining method. In this method the cells are labeled with Bromodeoxyuridine, a thymidine analogue by growing them in the solution containing Bromodeoxyuridine. The staining is done by initial exposure of the chromosomes to fluorochrome, irradiation using ultra violet light and then staining with the Giemsa stain.
The Ames method is less time consuming where the results are obtained within 48 hours whereas the test methods using cell lines to detect the Genotoxicity of a compound takes about 21 days to get the result. This is because of the slow growth rate of the cell lines compared to that of the bacteria. Also methods using cell lines should adopt sophisticated techniques to maintain the cell line.
The basic principle of genetic engineering is gene transfer, achieved by various methods to produce recombinant proteins, genetically modified microorganisms, transgenic plants and transgenic animals for commercial application. Genetic engineering, thus ultimately influences the growth of biotech industry. The two significant feature of genetic engineering is production of beneficial proteins and enzymes in surplus quantities and creation of transgenic plants, transgenic animals and genetically modified microorganisms with new characters beneficial for themselves using recombinant DNA technology. The discovery of a new protein either with a therapeutic property or application in food industry by a researcher or scientist would not have reached humans, for the use by humans without the application of genetic engineering in mass producing such proteins.
Recombinant proteins production and uses: The industrial production of proteins is done by transferring the desired gene responsible for the particular protein to be manufactured from the source organism to the preferred host organism through recombinant DNA technology. The host organism can be a bacteria or a eukaryote. The most preferred bacterial host is Escherichia coli for industrial production of proteins. The well established gene structure, faster growth rate, easy to cultivate and handle are the salient features of the E. coli bacterium fascinated the bio technologists to use this in recombinant protein production. Besides all these commendable characters of E. coli, the final output product is found to be unstable and difficult to purify. As a result research encouraged the use of eukaryotic host like yeast, cells of insects and cells of mammals in protein production. The proteins produced in this way find its way into pharmaceutical industry and food industry.
The recombinant proteins produced in the industry using the techniques of genetic engineering acts as drugs for various human diseases. To name a few, insulin produced for diabetes, alpha 1- antitrypsin in treating emphysema, calcitonin to treat rickets, interferon to treat viral infections and cancer, Factor VIII for hemophilia, production of growth hormone to act against growth retardation and chorionic gonadotrophin in the treatment of infertility. Some of the industrial manufactured enzymes occupy a vital position in the food industry. For example, the recombinant enzymes like rennin and lipase are used in cheese making, the role of alpha- amylase in beer industry, the antioxidant property of the industrially produced enzyme catalase and the use of protease in detergents.
Uses of Transgenic plants: In order to improve the quality and quantity of plants, traditional method of plant breeding is replaced by the creation of transgenic plants. The transgenic plants are plants carrying foreign genes introduced deliberately into them to develop a new character useful for the plant. The infection of plants by microorganism mostly viruses, poor production and decline in quality of plants due to attack by insects and the plants inability to withstand the pesticide or the weedicide used in the agriculture process welcomed the genetic engineering technology to develop transgenic plants with new characters like resistance to infections, defensive against the attacking insects and resistance to pesticides or weedicide.
The transfer of gene responsible for the protein protoxin from Bacillus thuringiensis to plants to develop resistance against the attacking insects is a remarkable example. Also the digestive action of the insects on the plants is restricted or inhibited by transfer of gene responsible for a particular protein with the property to arrest protease activity. The pesticides and weedicides used to destroy the pests and weeds is also a threat to the cultivated plants. The effects of such chemicals are alleviated by developing a new character called resistance to chemicals in plants. Development of resistance in plants against the weedicide glyphosate states the role of genetic engineering in plant breeding.
Uses Transgenic animals: Transgenic animals are animals carrying foreign genes deliberately introduced into them and exhibiting the characteristics of the introduced gene. Animals are suitable for various research activities trying to help mankind. In that way transgenic animals are created to study human diseases to derive appropriate treatment methods and to develop and identify the drug useful to treat the disease. The presence of human proteins in milk of animals is made possible by genetic engineering. Gene transfer is done in animals to increase the milk production and to increase the growth.
Like a coin has two sides, the other face of genetic engineering like creation of genetically modified organisms to be used as biological weapons is not welcoming.
The best outcome of the Recombinant DNA Technology is Transgene. The Transgenic Plants or Genetically Modified Crops are the plants produced by having certain genes transferred from another species into that particular plant through various natural or artificial insertion techniques. This conventional technique goes handy and very precise in comparison to time consuming classical methods of breeding. The noble causes for producing transgenic plants are many; like enhancing the agricultural values (quality), for higher yields, for manufacturing certain commercially important products like pharmaceutical products, proteins; and also importantly to carry out the detailed study about various physiological and biological processes during the plant’s development. Tobacco was the first transgenic crop, in 1983, expressing the Kanamycin resistance. Well known Flavr Savr tomato was the transgenic plant to be first commercially launched in the market. Then started the never ending research and production of various transgenic plants and crops’ line expressing various biotic and abiotic stresses resistances and more.
Transgenic plants have both the commercial and applied benefits, which includes introduction of herbicide resistant gene, virus resistant genes, genes for self-incompatibility, pigmentation in floral products and tolerance to biotic and abiotic stress. Also as vaccines for immunization against various pathogens. As tools for studying plant molecular biology, mutations, etc. these GM plants have been aiming to produce various immunoglobulin, interferon and some useful polymers as well. The applications and the methods goes hand in hand to understand the GM plants. We shall discuss it in the paragraphs below. The method plays a very significant role in producing the transgenic plants and the foremost crucial part for production of the transgenes are construction of the gene and the gene transfer method. Plant gene in general comprises of regions: promoter, enhancer, cap site, leader sequence, initiation codon and a stop codon, exons and introns, an untranslated region and poly A tail. Each of these regions have different role and these specification signifies the assembly of DNA sequence designing and its expression in the transgenes.
The methods for gene transfer are categorized into two types: Vector Mediated gene transfer and Direct Transfer i.e., vector-less gene transfer method.
First one, Vector Mediated, as the name suggests is carried out by plant viruses being the vector or by Agrobacterium mediated transformation. Agrobacterium tumefaciens are considered the natural genetic engineer which conveniently infects any plant tissue or organ ensuring the large fragments of DNA with reasonably good stability and effective regeneration capability. Plant viruses are natural vector for genetic engineering and they can efficiently introduce the desirable genes into almost all plant cells systematically. The plant viruses are majorly inserted into a plant chromosome.
Second one i.e., Direct (vector-less) DNA transfer allows the foreign DNA to directly insert into the plant genome. These methods are more simple and effective. These methods includes: DNA absorption by cells/ tissues, physical gene transfer method and chemical gene transfer method. DNA absorption has very little or no success rate, but it is believed that the DNA gets absorbed and the cells get transformed in the cells suspensions. The physical gene transfer method includes: electroporation technique, gene gun (particle bombardment), microinjection, liposome fusion and silicon carbide fibres. The chemical gene transfer method includes: polyethylene glycol mediated and diethylaminoethyl dextran mediated transfer method. In addition to methods described here, there is one more new transformation technique known as chloroplast transformation which is in the developing stages and it holds efficiently promising future in plant biotechnology.
After the transformation of plants is accomplished, they need to be confirmed for being transgene by various selecting tools. These tools are set of genes referred as marker genes: selectable marker genes (eg. Bleomycin resistance genes, β-glucoronidase, Acetolactase synthase) and reporter genes (eg. Greeen fluorescent protein, Luciferase). Further step is to study and ensure the expression of genes which is carried out by promoters and terminators. Next step involves confirmation of integration of transgenes with the targeted plant genome; this is confirmed by techniques- southern hybridization and polymerase chain reaction. Later comes ensuring of transgene being stable in terms to avoid gene silencing followed by regeneration of the transformed plants which are transgenic plants.
These all genetic manipulations are done with the aim of improving crops with desired traits such as biotic and abiotic stress resistant, quality and yield, enhancing the nutrition and use of genetically modified plants as bioreactors. Biotic stress resistant crops include:
(i) Pest resistance by the use of Bacillus thuringieneses (Bt) toxins producing wide range of cry proteins; protease, lectins, etc. (Bt crops includes Bt cotton, rice, maize, tobacco, tomato, potato, cowpea and soybeans),
(ii) Virus resistance by incorporating virus coat proteins, antisense RNA technology, ribosome, etc. and
(iii) Fungal and bacterial disease resistance by incorporating pathogenesis related proteins, phytoalexins. While, abiotic stress resistant plants includes: herbicide resistant, drought and soil salinity resistant, freeze resistant, etc. Genetic modification however has contributed tremendously in the crop yield and quality; such as extended shelf life, slow ripening, and preventing discoloration in flowers, fruits and vegetables. Transgenic plants with improved nutrition have been engineered for human health improvement, e.g. Golden rice which is enriched with provitamin A, potato with increased protein and methionine levels, canola with cis-Stearates-lowering the risk of heart diseases, sugar beet with fructans-low calorie alternatives to sucrose. Also, transgenic have been engineered for allergen absence. Most importantly, transgenic plants as bioreactors have created boom in commercial aspect for manufacturing hemoglobin, monoclonal antibodies, interferons, serum albumin, proinsulin, edible vaccines, etc.
Despite of all these advantages the GM crops/ plants have been much controversial from ethical point of view. Nevertheless, GM crops have been potentially engineered in an eco-friendly way causing improvement to human health and environment.
Gene cloning involves application of many enzymes having specific functions to result in a modified product. Of the different enzymes involved, endonucleases are a type of enzymes which has the ability to cut or cleave the DNA molecule. Restriction endonuclease refers to a group of endonucleases which cleaves the DNA at specific points known as recognition sequences or sites. Different types of restriction endonucleases have been identified like type I, II, and III. Among these, the most available and most extensively used enzyme is type II restriction endonuclease. Examples are: ECoR I, Hind III, etc.
The importance of restriction enzymes lies in the property that it cleaves the DNA sequence, in most cases, within their specific recognition sequences unlike other restriction enzymes which cuts some base pairs away from their recognition sites. Some type II restriction endonucleases are also known to cleave the DNA sequence in close proximity of their recognition sequence rather than within the recognition site. This efficient nature of type II restriction endonucleases, combined with their comparatively smaller structure, has led to the wide application of these enzymes in gene cloning.
Mechanism of type II restriction endonucleases.
Recognition sites are specific area or sequences in the genetic molecule which these enzymes recognize as sites for cleavage. Recognition sites are unique for different restriction endonucleases. For type II restriction endonucleases, recognition sites are mostly palindromic sequences with rotational symmetry. DNA has a double stranded helical structure where, the nucleotides of the two strands of DNA are complementary to each other. There are certain sequences in such a structure where, the first half of the sequence is a mirror image of the second half of the complementary strand and reads identical from same end. Such sequences are termed as palindromic sequence with rotational symmetry.
The restriction endonuclease moves along the surface of the DNA until it recognises its target sites. After recognition, it initiates DNA binding in the presence of Mg2+ ions resulting in cleavage at specific sites.
The cleavage patterns produced by different restriction endonucleases are specific and each holds a novel role in gene cloning.
The two main patterns of cleavage are creating staggered cuts and even cuts. In staggered cuts, the cleavage occurs in different locations resulting in producing protruding ends of one of the strands in the double helix. Such ends are known as cohesive or sticky ends. The main benefit of such ends is that the protruding ends created are usually complementary in nature and can be used to link with vectors consisting complementary sequences for isolating the DNA fragment. It forms the basics for recombinant DNA techniques such as southern blotting. The even cuts, on the other hand, produces blunt ends where the two strands are cleaved at similar points. The importance of blunt ends in gene cloning involves many techniques which are utilized to modify the blunt ends in a manner so as to meet the specific requirements.
Tailing: This is a procedure which results in a protruding end of a defined length being created which aids in the pairing of required DNA segment with appropriate vector.
Linker: Linkers are chemically synthesized oligonucleotides. This can be used to modify the blunt ends so as to create cohesive ends of required bases. Linkers are so designed as to have a recognition site of a specific endonuclease. This can be linked to a blunt end DNA fragment created by the restriction digest. Such a modified fragment when digested by the linker specific restriction endonuclease can create cohesive ends complementary to vectors which can later be isolated to create multiple copies or, can be used in creating a recombinant DNA.
Adapters: These are short artificially synthesized double stranded fragments which can be used to link two blunt ends with different end sequence.
As a result of all these techniques, it is possible to alter a specific gene at a nucleotide level by identifying the respective restriction endonuclease enzyme which can cleave at the specific site. This is the principle adapted in gene cloning given that, to create a specific clone it is required to isolate the target gene. This isolation can only be done with the help of a restriction endonuclease enzyme. The type II restriction enzymes accentuates the importance as they have the ability to cleave at exact points resulting in producing definite fragments rather than random fragments.
(i) RFLP (restricted fragment length polymorphism), which involves production of DNA fragments of different lengths which can be separated and utilized for several purposes like DNA fingerprinting, identification of mutations, preparation of genomic library etc.
(ii) A technique called restriction mapping make use of the capability of restriction enzymes to create DNA fragments of specific length thus distinguish alleles of a single gene having altered restriction sites.
(iii) Gene therapy: This employs the property of restriction endonuclease to recognize and remove a specific DNA fragment responsible for many diseases.
There are certain disorders like cancer, parasitic and viral infections, which cause excessive production of specific proteins in day to day life. An alternative treatment for these disorders is known as Antisense Technology. A single stranded RNA that is complementary to messenger RNA (mRNA) is referred to as Antisense RNA. The antisense therapy includes inhibition of translation by using single stranded nucleotide, any DNA or RNA sequences or even synthetic ones. From the practical point of view, most of the antisense therapies work efficiently and produce best results if used with RNA since RNA specifically binds to target mRNA and blocks protein synthesis.
Antisense technology was also referred as Gene Subtraction, but it is proven to be a misnomer as this technology does not remove gene, rather it just involves inactivation of the gene. Naturally occurring mRNA antisense mechanism is the hok/sok system in E.Coli R1 plasmid.
The antisense technology is carried out on the basis of the principle that the cloned gene is ligated into the vector in reverse orientation. Now, as the antisense technology obstructs the mechanism of translation it is stoichiometric in nature, and it can prevent synthesis of the product of the gene it is directed against. The antisense RNA mechanism involves hybridization of the antisense and sense copies of RNA. Now, as the ds-RNA molecule is formed, it is rapidly degraded by ribonucleases and the expression is blocked. Another reason could also be the antisense RNA preventing ribosomes getting attached to the sense strand. In simpler words, if an oligonucleotide is introduced into the cell, it binds to specific mRNA which forms an RNA dimer in the cytoplasm and halts the translation mechanism; this is because the mRNA no longer has access to ribosome and dimeric RNA is rapidly degraded by ribonuclease which in turn on introduction of oligonucleotide complementary to mRNA leads to blockage of translation by particular gene, turning off the gene.
RNAi (RNA interference) and Antisense RNA technology though has the same effect but their mechanisms are quite different. Firstly, RNAi technology involves degrading mRNA by small interfering RNAs triggering catalytic gene silencing; whereas in Antisense RNA technology mRNA is degraded by RNase H. secondly, in comparison to antisense RNA, RNAi are twice larger.
The application of Antisense RNA technology is in many sectors. This technology was used in Flavr Savr, for tomato ripening; ripening in tomato produces enzyme Polygalactourodase (PG) which softens tomatoes and finally rotten them quickly. Two biotechnology companies: Calgene, USA and ICI Seeds, UK introduced a gene in plant which synthesized a complementary mRNA to PG gene and inhibited the synthesis of PG enzyme delaying over ripening and rotting. Antisense therapy is considered for treating certain genetic disorders and infections. This is also referred to as Gene Therapy. It includes: isolation of specific gene; it’s cloning and inserting it into target tissue cells to make desired protein. It has to be ensured that the gene is expressed correctly and sufficiently without causing harm to patient in context with the immune response. Antisense RNA technology is also used for cancer therapy. This therapy was used for treating brain cancer – malignant glioma and cancer of prostate gland, in malignant glioma IGF-I was over produced and in the prostate cancer IGF-IR was synthesized more. These two were used to block the translation. Research is carried on in regard with Antisense RNA drugs for treatment of CMV, HIV, cancer, etc. Antisense antiviral drug named Formivirsen is developed to treat CMV, which was licensed by FDA in August 1998. In 2010, scientist at NIPGR by using Antisense technology developed tomato which could last longer for more than 30 days by silencing two genes (alpha - man and beta – hex) which causes softening and wrinkling in tomatoes during ripening process.
Despite of achieving some success, Antisense technology has few challenges. Like: delivery into the patients body, then possibility of toxic effects due to it’s regulation on both the normal and mutant alleles. Antisense RNA technology is also used to study certain gene functions.
Looking at the advancements in the Antisense RNA technology, it could lead to potential development of pharmacological agents, studying physiological and pathological processes as well as it’s use in effective treatment as gene therapy.
Advances in the field of biotechnology, research and medicine has made it possible to produce artificial skin in vitro.
A culture refers to culturing of any kind of cells or growth of cells on a suitable nutrient medium under in vitro conditions. A cell culture initiated to formation of tissues can be termed as tissue culture. It is basically of two types:
(i) Cell culture: where the tissues which are cultured are broken down into cells by enzymatic or mechanical means.
(ii) Organ culture: where the tissue culture are further developed into maintaining their structure and resulting in production of specific organs.
Isolation of animal cells and establishment of its successful cell culture leads to formation of primary culture. This when sub cultured produces cell lines. Cell lines which dies after several subcultures is known as finite and those which survive multiple subcultures and continue to grow indefinitely is known as continuous cell line.
Culturing of animal cells under invitro conditions leading to production of organs or parts of organs such that, they retain or closely exhibits their structure and functions is known as organ culture.
Human skin is the largest organ of the human body giving structure to the body and serving as the first line of defense of the body against infections. It is basically formed of two layers: the outer or epidermal and inner or dermal layer.
Advances in the field of biotechnology, research and medicine has made it possible to produce artificial skin in vitro. The first successful result in this field was accomplished in around 1970s by two scientists named Burke and Yannas. The synthetic skin they produced was termed as Silastic. The Silastic so produced was successful in producing only the epidermal layer of skin and could not, however, develop the dermal layer.
Living skin equivalent (LSE):
A further development led to production of structure called living skin equivalent which essentially resembles complete skin (epidermis as well as dermis).This is also known as graftskin.
The corresponding procedure involves isolation of living skin explants either from the required patients or from the newborn infants; culture and growth of such explants in a collagen matrix.
The skin at large is made up of cells known as keratinocytes. These cells produce dead cells or corneocytes which make up the outermost layer of skin. The keratinocytes during their transition to corneocytes expel lipid molecules. These dead cells along with lipid molecules together form the living skin equivalent under invitro conditions. The explant isolated consists of keratinocytes extensively. This undergoes trypsin treatment in order to break down the tissue into cells. Studies have revealed that irradiated 3T3 fibroblasts, which is a continuous and non tumorigenic cell line, promotes keratinocyte growth and proliferation. Thus, following the trypsin treatment the cells are cultured in 3T3 lined vessels. The proliferation result in the formation of colonies of cells which is again subjected to dissociation into individual cells. After a number of cultures and subcultures, a pure multilayered sheet of epithelium called cultured epidermal sheets (CES) is achieved. These sheets produced are then detached from culture vessels, cleaned and then applied for grafting. For the complete success of the procedure, it is important that the living explants are isolated from the respective patient so as to defer the possibility of rejection by the patient’s immune system.
(i) Treatment of burns: - The most important application in developing a living skin equivalent is in the treatment of people suffering severe burns. Often in such cases when grafted with the patient’s own skin taken from elsewhere in the body, it does not regenerate rapidly to cure the burns effectively. Similarly, xenografting can induce severe rejection reaction from the body. But the graftskin technology has proved successfully to aid in such treatment to larger extent so much so that, majority of the fundamental skin components were regenerated. This is also of great importance to people affected with skin cancer wherein such graft skin can be used for developing non cancerous cells and thus aid in cancer treatment.
(ii) Wound healing: - Another vital role of graftskin is in healing of wounds produced on skin by various diseased conditions. Thus, chronic skin ulcers (eg: foot ulcers developed as a result of diabetes) can be treated with living skin equivalent.
(iii) Providing model environment for research: - Since the graftskin produced resemble the living skin extensively, it can be employed as a medium to carry out various skin related research.Variations in the medium used for developing the related tissue has been introduced such as, introduction of fibroblasts for better differentiation and vitamin c for improved barrier functions of differentiating cells. Diseased skin condition can also be induced under invitro conditions in such living skin equivalents so that they can be studied closely into developing probable cure.
(iv) Testing dermatological products: - Artificial skin can also be used to test the effectiveness of various dermatological products rather than testing on the lab animals.
Thus it can be concluded that the living skin equivalent or artificial skin exhibits a wide range of future prospects having clinical, laboratorial and medical applications.
DNA Microarrays consist of a number of DNA spots which are attached to solid substrates like glass, silicone, nylon membranes.
After a gene has been isolated, the next step involved is to study the different characteristics of the isolated gene. In the past, it was possible to analyse the nature and properties of only a single gene but with the advent of microarray technology it is possible to analyse thousands of gene with a single experiment resulting in a faster and more accurate results.
Microarray: It refers to a recent hybridization technique which provides an opportunity to match known and unknown DNA samples under specific conditions to reveal different properties of the unknown gene. Here, a series of probes is fixed onto a solid substrate and used to hybridize a series of test DNA. It is of two types DNA microarray and antibody microarrays.
DNA microarrays (DNA chips, bio chip, gene chip):
This consists of a number of DNA spots which are attached to solid substrates like glass, silicone, nylon membranes etc. In a more recent technique, an array of microscopic beads is used as a platform for such arrays.
Probe and Target:
A probe refers to a small nucleotide sequences with known bases. This is fixed onto a solid substrate to form a neat arrangement called an array. Purified mRNA, isolated DNA, cDNA produced from mRNA; all can form probes.
Target refers to test a DNA sequence which has to be studied for its position, composition, gene expression, mutations etc; these are labelled using fluorochrome or other labels which aid in quantifying the results after hybridization.
DNA microarrays are of two different types
The probes consisting of complementary DNA is spotted onto the glass substrate with the help of fine needles and a robotic arm.
The probes here are oligonucleotides formulated in situ or produced externally and then immobilized on the arrays by different techniques like photolithography.
Earlier the oligonucleotide array was termed as DNA chips. But recently the term DNA or gene chips are applicable to both the kind of arrays.
Principle and mechanism:
In order to conduct such a study, the complementary base pairing nature of the polynucleotide chain of the DNA and the resulting double helix plays an important role. In the process of hybridization, these polynucleotides are subjected to conditions so that the hydrogen bonds between the two stands weaken but phoshodiester bonds between the nucleotides remains intact leading to production of two separate polynucleotide strands. When treated with appropriate probes, it results in the pairing of the probe with its complementary bases in the target DNA.
The DNA sequences of each gene to be analysed of an organism are labelled with fluorochrome like Cys green or red. DNA micro array is set up by spotting or other available techniques. When such a probe is treated with the labelled target DNA and hybridization is initiated, the double helix open up and hybridize with complementary DNA resulting in producing fluorescence at hybridized sites. These are scanned and quantified. Different fluorochrome representing different properties can be used to be probed with a single DNA chip resulting in conducting multiple tests using a single microarray.
Multiple and single channel microarrays:
In a multiple channel or two colour microarray, it is possible to analyse genes from different sample in a single test. Each target gene is labelled with fluorochrome having different fluorescence emission. This mixed sample is allowed to hybridize in a single test with a single microarray probe, and when scanned with microarray scanner after excitement with different corresponding wavelengths, the ratio of different gene can be quantified.
In a single channel or one colour array, a probe is hybridized with target DNA labelled with one colour fluorochrome. Two colour microarrays is usually used to detect different genes present in one mixed sample whereas one colour microarray usually is employed to estimate the amount of gene expression of same sample or between samples. Single channel microarrays are found to give more accurate results but need to conduct different test to quantify different gene expression. Multi channel makes use of only one test to give multiple results but different samples sometimes interfere with each other leading to non unique results.
Micro array technology can be used to detect presence of genetic diseases, presence of mutations, polymorphism, cancer etc.
(ii)Gene expression profiling:
Using this technique, the number of genes present, the expression rate each gene can be determined. Monitoring the gene expression is known as gene expression profiling. Quantitative and qualitative measurements of such expressions is possible with the help of microarrays.
With the help of microarrays, SNPs or single nucleotide polymorphisms can be detected. It refers to different nucleotides present at the same base position in different alleles. Such minute differences between different individuals or different alleles of same gene can be identified successfully and uniquely with the help of micro arrays.
A microarray is prepared with oligonucleotides of specific length. When hybridized with target sequences of unknown lengths, the results obtained indicate the length of the target sequence.
The gene expression is determined by two features called as penetrance and expressivity of the genes. Penetrance is the ratio of individuals exhibiting expected phenotype and expressivity is the extent of gene expression in an individual. The phenotype of an individual is determined by the genotype or the type of gene expressed. In general, phenotypic changes occur in individuals when exposed to various environmental factors. But the query, “Is the genotype of an individual is influenced by external environment?” lead to several researches throwing light on the effect of external or environmental factors like temperature, light, chemicals and nutrition in gene expression. Besides the effect of internal factors like hormones and metabolism on gene expression, external factors were also found to affect the gene expression and ultimately exhibiting phenotypic changes.
Temperature and Gene Expression: The study on the coat color of the Himalayan rabbit with relation to the temperature reveals the effect of temperature. The rabbit with normal phenotype of white fur showed differences in the skin color with exposure to temperature. The body of the rabbit which is generally exposed to high temperature (>34 degrees) expressed white color whereas the other parts like ears, nose , tail and paws which are little exposed to temperature expressed black color. Keeping the rabbit under cold climate resulted in the expression of fully black colored skin. This study proves the sensitivity of the genes responsible for the skin color to the temperature.
Another study on wing development in Drosophila flies with response to the temperature also provided results with the effect of temperature. Flies exposed to the temperature of 25 degree Celsius showed less penetrance whereas when exposed to higher temperature penetrance also increased which was observed by the increase in the development of wings in the selected population of flies.
The research by the scientist Voolstra CR and his team from KAUST, Saudi Arabia on studying the gene expression by exposing the embryos of Coral Montastraea faveolata to different temperatures like 27.5, 29 and 31.5 degree C resulted in the continuous expression of stress related genes in the embryos that were exposed to 31.5 degree C. Also the effect of temperature on genes encoding the enzyme for the biosynthesis of starch in the wheat plant Triticum aestivum was studied by William J Hurkman and his co-workers from USDA, USA. The effect of temperature on these genes was observed by analyzing the starch accumulation.
Also the research on the effect of temperature reduction on gene expression and oxidative stress in skeletal muscle from adult Zebra fish by Ranae L and team and the study of sea water acidification and elevated temperature’s effect on gene expression pattern of the Pearl Oyster Pinctada fucata by Wenguang Liu and team, China shows the role of temperature, an external environmental factor on gene expression.
Light and Gene Expression: The study of a gene responsible for the anthocyanin pigment formation in Maize plant with relation to the light by researchers is a good example showing the role of light in gene expression. The plant carrying the homozygous gene for pigmentation when exposed to sun light developed bright red color and when the light was retarded by covering the area of the plant prone to pigmentation, the bright red phenotype was not observed. Also the prevalence of skin cancer in humans on exposure to sunlight is a classic example.
Chemicals and gene expression: The research by Mankame T and team from Texas University on a fungicide called Enable which has potential effect on endocrine regulated gene showed down regulation of 8 genes and upregulation of 34 genes on exposure to the chemical. The effect of chemical mutagens and carcinogens on gene expression profiles in human TK6 cells by the researcher Lode Godderis and his team developed results. In their experiment, they observed a linear trend in Dose- response of gene expression for chemicals like Trichloroethylene, Benz(a)anthracene, epichlorohydrine, benzene and hydroquinone. The effect of the sedative drug Thalidomide on fetus causing birth defects can also be coined as an example for the effect of chemicals.
Nutrition and Gene Expression: It is a very interesting fact that dietary and nutritional supplement also plays an important role in gene expression. The deficient nutrient supplement alters the genetic expression. What a pregnant woman eats determines the health of her offspring. Babies born with deformities to mother ingested with Thalidomide drug during 6th week of pregnancy in 1960s should be cited. The folic acid supplement to the pregnant women takes care of the development of the fetus whereas the deficiency of the same causes some birth abnormalities.
Thus the fact that the environmental factors also play a vital role in gene expression is understood by various research studies.