CURRENT USES OF GENETIC ENGINEERING

DNA fingerprintprotein bands

a) DNA FINGERPRINTS

--Restriction endonucleases are enzymes that cut nucleic acid chains (bacteria use them as a defense against viruses). These cuts aren't random; they occur at only specific sequences. Since these cuts are constant in this way, 2 individuals can have their DNA molecules cut and patterns between the individuals (the degree of similarity/ dissimilarity) can be seen after sorting and staining the DNA fragments through a process called electrophoresis. This is especially useful with parts of the human genome known as RFLPs (restriction fragment length polymorphisms), areas which, unlike most of the genome, can vary greatly between individuals.
APPLICATIONS:
1) Diagnose genetic diseases in individuals, even the unborn.
--This may give indication of risk.
--In half of all heart disease patients, the first symptom of a problem is heart attack and death; obviously some degree of testing before symptoms are evident can be useful.
--All armed services personnel are tested for being carriers to sickle cell anemia; many are unaware (since this has no effects in normal conditions) but can cause death in extreme stress or in flight related exercises.
--Identifying those at risk early may lead to preventative measures (earlier and more frequent mammograms for those at risk for breast cancer) or provide test groups willing to try experimental treatments in time to prevent symptoms.
EXAMPLE

--Gene profiling systems to diagnose have been patented; one involves 2500 genes.
--Fingerprinting mitochondrial DNA from saliva and urine may give some warning of cancer (mitochondrial mutations may help cancer cells be more active).
--Removing fetal cells through amniocentesis of chorionic villi sampling provide fetal cells for testing. This is typically done when parents consider terminating the pregnancy depending on the result; this may lead to treatments in the unborn: Caesarian births increase survival in infants suffering from myelomeningitis, infants with PKU are placed on special diets to prevent severe retardation, periodic blood removal prevents hemochromatosis.
2) Identify criminals on the basis of blood or semen left at a crime. This was used for the first time in England in 1986 (after being used in an immigration case the year before) to clear the prime suspect of a crime, it was used later that year in Florida. It has since been used to decide thousands of cases . A few states, such as Louisiana, perform DNA fingerprints on all convicted criminals while many states perform fingerprints on all those convicted of certain crimes (e.g. in 1999, the state of New York increased the number of such crimes to over 100 which will probably increase the number of tests from 3,000/yr to over 30,000; these new crimes include burglary, arson, and drug dealing). Over 50 individuals who were previously incarcerated have been exonerated through DNA fingerprinting. More than 25 countries use this procedure.
EXAMPLE:

--As of the year 2000, states had collected 600,000 samples but only 250,000 had been analyzed. The FBI database contained 138,000 samples.
In one case, the DNA fingerprinting of pollen on an individual's clothes proved he was at the crime scene and led to a confession.
--because crime scenes often contain minute amounts of DNA, a technique known as PCR is employed in which DNA is heated to separate the strands after which a heat stable DNA polymerase copies each strand; this process can be repeated as often as needed
--other individuals can also be identified; one unknown soldier was identified
3) Decide paternity questions. Today, only about 0.1% all paternity cases go to court. This technique has solved immigration questions and has reunited 50 (of 200) Argentinian children whose parents died in the 1975-83 political purge (9,000 vanished and were later found dead) have been reunited with their biological relatives.
EXAMPLE
In the example below, both the mother and the father will have DNA pieces which are not present in the child (remember that sperm and ova only carry half of an individual's chromosomes, not all of them). To determine which of the two males is the father of the child, you need to look for which could provide the DNA fragments present in the child which were not present in the mother.

4) Distinguishing between different strains of microbes. Strains which are virtually identical in almost all respects can vary in their virulence (as in normal E. coli and E. coli 0157); this may be the only way of distinguishing between them and will become more and more important with the evolution of antibiotic resistant strains.
5) Work with animals. Measuring the degree of relation can minimize inbreeding and is used in anything from domestic chicken to endangered species. Poachers of hyacinth macaws were convicted when it was proved they were not breeding them. DNA from restaurant and market samples can be tested to determine if endangered species have illegally been killed. Dog pedigrees can be determined. Pure research is often served: it has been shown that many "monogamous" birds have offspring of mixed parentage.

6) Evolutionary relationships can be determined by comparing samples from different groups and sorting them on the basis of similarity. This has given the strongest support to evolutionary theory. DNA is sometimes preserved in fossils and can be studied (so far from fossil magnolias, a cypress, insects, mammoths, a tasmanian wolf, and a neanderthal).
7) Endonucleases specific to each of the 4 DNA bases are being used to determine the sequence of human DNA. This will take some time to determine: if you could print 2,400 bases on a page, you would need 3,000 books that have 1,000 pages each to describe our entire genome.

b) INSERTING DNA FROM ONE SOURCE INTO ANOTHER

bacteria

The bacteria in the dark colony above and the surrounding lighter colonies are transgenic organisms (also called genetically modified organisms or GMOs). They possess genes from two different sources. Although the source of the added DNA in this case was another bacterium, it could just as easily come from a plant or an animal (including humans). This process is not necessarily "unnatural" in that the bacteria in your large intestine might absorb DNA from their environment whether it be from dead bacteria or from dead human cells.
Enzymes called endonucleases can cut a gene from one source's DNA and the ligase could add it to another source, often a plasmid. Plasmids can introduce new genes into bacteria and plasmids inside viral particles (called cosmids if they have two cos sites for packaging) can introduce new genes into plant and animal cells (including humans). Very large DNA pieces are incorporated into yeast artificial chromosomes (YACs) which behave as natural chromosomes inside eukaryotic cells. In 1971, DNA from different sources was mixed for the first time; in 1978 the mammalian protein was produced in a transgenic bacteria. This can be performed through transformation, microinjection (human genes were first injected into mice in the 1980s), plant tumor viruses, viral infection (there is a size limit to the DNA which can fitBthe CFTR gene can=t fit but its cDNA can), receptor mediated, liposomes (lipid membranes that surround plasmids), electric currents (which increase the uptake of foreign DNA in eukaryotic cells), somatic cell embryogenesis, and embryonic stem cell work. Complimentary DNA can be used to target specific genes to inactivate (knock-out).
APPLICATIONS:
1) Bacteria have been given new genes and now make new molecules. Human genes in bacteria produce molecules important for our bodies like insulin (which was fomerly collected from slaughtered cattle), growth hormone (which was formerly collected from cadavers), interferon (collected from urine), erythropoetin, interleukin, alpha antitrypsin (to treat emphysema), NGF, and tPA. Other bacteria have been engineered to produce antigens necessary for a hepatitis B virus (as well as several animal diseases), rennin (important in cheesemaking) and phenylalanine (necessary for the manufacture of Nutrasweet). Some microbes have been engineered to release hydrogen gas or ethanol for fuel, synthesize biodegradable plastics, metabolize toxic compounds, sequester heavy metals, metabolize sulfur (and perhaps reducing the severity of acid rain), improve beer quality (both German and Japanese brewers have altered yeast strains), combat disease in the wild (bacteria that live in the arthropod that can carry Chaga=s disease were released that produce peptides that may kill the disease), help clean oil spills (this was the first patented microorganism), and help loosen oil to aid recovery.

2) New DNA added to plants to increase their agricultural yield. This is currently a billion dollar industry with over 50 crop varieties available (you have already used engineered products; 1/3 of U.S. soy crop); occupy over 76 million acres of U.S. cropland. Some produce specialty oils for cosmetics & shampoos, oils (rapeseed, soy, and sunflower) to decrease dependency on foreign oils, produce cooking oils lower in saturated fat, resistant to biodegradable pesticides, resistant to pesticides in general so that more weeds can be killed, resistant to viruses (which damage millions of dollars of crops/year: melons, tomatoes, squash, cucumbers, potatoes, alfalfa, tobacco), frost resistance, tomatoes that soften more slowly so that they can ripen on the vine (2 varieties; 1994 FDA approved Flavr Savr was first commercially available product), blue green algae engineered to produce eicosapentaenoic acid, a health food ingredient previously obtained from fish, engineered flowers bloom longer, engineered flowers with stripes and swirls (even different colorsBblue carnations), better-smelling flowers (current breeding often eliminates smell, engineering is improving yields of biofuels; cottonwood stands have increased yields of 3-4x, grapevines resistant to stem-sucking insects, tomatoes with the antioxident lycopene which decreases cancer risk, beans with less carbohydrates which cause less gas, soy with more isoflavones which decrease heart disease risk, sweet peppers which are sweeter, lupin with improved digestibility & nutritive value, potato strain that can kill the Colorado potato beetle with its own insecticide, barley that can be irrigated by salt water, and Bt corn which makes its own insecticide (which has been manually used by farmers for 30 years). There are many other varieties ( zucchini, soy, watermelon, squash, potatoes that have more starch for better-tasting chips which absorb less oil in french fries, and naturally decaffeinated coffee beans) which are currently awaiting approval. Over 2000 field trials have been performed. Due to the absence of deleterious effects, the USDA in 1993 relaxed its restrictions on engineered plants. Other plants have been engineered to produce materials for vaccines: soybeans & genital herpes, potatoes for diabetes & cholera, tobacco and potato make enterotoxin (mice which eat develop antibodies against the toxin; same seen in humans), and current work on rabies (tomatoes & spinach) and oral measles (tobacco) vaccines. Some plants have been engineered to make fructans for low calorie foods, phytase which enhances phosphorus utilization in cattle, beta-glucocerebrosidase to treat Gaucher's disease.
--Some argue that adding new proteins to foods can cause allergic reactions from people who feel that a certain food is safe for them to eat. As a result GMOs may be required to undergo extensive testing: one new product underwent 1800 tests while many new strains generated by more conventional methods undergo no testing at all.
--In the U.S. 20-45% corn and soy now produce their own pesticides, reducing the amount which is sprayed. There has already been an 80% reduction in the amount of pesticide sprayed on cotton.
3) Animals: Some substances are being added to milk (goatsmilk: anti-thrombin III, human monoclonal antibodies to treeat psoriasis, organ rejection, and autoimmune disorders; sheep: human clotting factor IX, albumin to treat burn victims, alpha-1 anti-trypsin for cystic fibrosis patients; cows: human lactalbumin, albumin; pigs: clotting factor VIII and fibrinogen), bovine sommatotropin from bacteria increase milk production, increasing resistance in strains will decrease the need for antibiotics in feed, hundreds of mutant mouse strains have been created to help in the study of human disease, research into engineering pigs whose organs (roughly the size of human organs) have human proteins so they can be transplanted into humans without immune rejection, more casein in cows milk for faster cheesemaking, chickens with less fat, chickens with less cholesterol in their eggs, anc transgenic fish and cattle which grow larger than normal or are meatier. The company Aquaculture have produced salmon, trout, flounder, and tilapia which grow to full size in half the time usually required.

4) GENE THERAPY (transgenic humans)
New DNA packaged in viral coats (adenovirus, retroviruses, HIV, lentivirus, Epstein Barr virus, Sendai virus), DNA packaged into liposomes, or cells which are transformed (microinjection, transformation, injection into a tumor, "gene guns" that use air pressure to shoot millions of tiny gold beads coated with DNA into cells) and returned to the body are the brightest hopes for the cure of genetic diseases. In 1992 3, 2 young girls and later 2 babies were given new genes to correct genetic deficiencies in their immune systems. There is research which is showing promise for the treatment of many diseases. Some procedures have been successfully used to give new traits to human cells in cell culture, others have been successful in animal studies and many have been used in humans. Here are some diseases and the status of research at this point. Some are being reviewed by USDA for approval, some may be given the fast track consideration.
Cystic fibrosis: CFTR has been introduced into cells through aeresol improves function but not a cure yet; recombinant Dnase I decreases DNA in sputum which contributes to its viscosity



cancer: increasing expression of tumor necrosis factors (TNF); increasing p53 tumor supressor protein suppresses melanoma, pancreatic cancer, colorectal cancer, and lung cancer; attaching suicide genes to other promoters and enhancers so that they are active in cancer cells, antisenseoligonucleotides to the oncogenes myc, myb, and ras have been shown to cancer proliferation, antisense c myc in prostate cancer; antisense bcr/abl used to fight leukemia, antisense oligonucleotides to 2 human papillomavirus oncogenes reduced growth rate, increased levels of cytokines and interieukin reduce cancer growth, interieukin 2 inhibits colon carcinoma, thyroid carcinoma, and renal carcinoma, brain tumors treated by producing a macrophage stimulating factor; interleukin and p53 used to treat breast cancer, integrin to fight melanoma, cytidine deaminase into hematopoeitic cells to fight blood cancers; pl6 and p2l used also in bladder cancer; inject tumor with DNA for a foreign antigen so that immune system fights (after drug induces); cytotoxic T cells engineered to befter recognize cancer, carboxyesterase CDNA for lung cancer, RBI for retinoblastoma; cells of removed tumors can be altered to release cytokines that attract immune cells and then reintroduced into patients, CD40 ligand aftracts immune cells; blocking Tie2 receptors can prevent tumor angiogenesis; down regulation of endothelial growth factor in glioma; thymidine kinase to treat pituitary adenomas and hepatocarcinoma; allovectin 7 for melanoma; vaccines made from the products of dendritic cells after being injected with cancer cell RNA; fusion of genes into leukemia cells in children lead to well tolerated chemotherapy; children with neuroblastoma treated; as of 2000 BRCA1 testing was about to begin
AIDS . mutant TRNA interacts with reverse transcriptase & interferes with early stages of the viral life cycle; the CD4 receptor which HIV viruses aftach to has been aftached to red blood cells (so that receiving this blood would decrease the amount of virus in the patient's blood stream), monocytes which have been engineered to express HIV antisense genes, and to antibodies; T cells from one individual (twins in this study) altered to attack HIV infected cells and injected into infected patient (cells lived longer than expected); gene gun of HIV gene products into Peyer's patches increases immune response; IFNA into leukemia cells; CD80 gene in cervical carcinoma cells increases response; interleukin 4 in glioblastoma
hemophilia: transfer genes which hemophiliacs lack (in mouse studies some stem cells in bone marrow were reached), now available in humans for hemophila B
STDs: plasmids introduced into vagina in rats increases IgA antibody production against antigen muscle loss with age: in mice, hormones introduced into cells retard muscle wasting
arthritis: CSK gene, anti inflammatory genes, interleukin 1, tumor necrosis factor
autoimmune diseases: engineered T cells for allergic encephalomyelitis
diabetes: B cells can be engineered to release insulin and returned to body
erectile dysfunction: cDNA injected into smooth muscle of rat penises
heart disease: expression of FGF and other growth factors help vascularization of heart
baldness: the protein beta catenin D in mice causes skin cells to develop into hair follicles
intelligence?: in 1999 the gene NR2B (which controls the production of the brain NMDA receptor,
with an important function in learning), created better learning miceBADoogie mice@; some
criticisms

Are these treatments safe? One patient died as a result of the virus with which he was injected; there is ongoing research to find the best strains of viruses to use. Another study was halted after two deaths which, although not a result from the treatment, were not reported properly.

 

CLONING
--the first cloning occurred in 1952 in frogs when tadpole cells were used to raise frogs
--another technology became more applicable to mammals: obtain a 4, 8, 16, 32, or even 64 cell embryo, separate the cells and produce that many distinct individuals
--after these early stages, cells begin to decide who they want to be when they grow up and turn certain genes off; these cells were no longer shown to be totipotent: able to produce an entire organism
--this animal work has many applications for animals that are especially good at producing milk, being meaty, racing, etc.
--in 1993, 8 cell human embryos were separated to form eight separate embryos (these human cells were abnormal to begin with and all embryos subsequently died); this was later done with normal monkey cells
--3/1997: a sheep and then a monkey were cloned from adult cells, which was previously deemed impossible
--this was achieved by starving cells and forcing them into inactivity