Lecture 15

Biotechnology & Genetic Engineering

Definitions

Biotechnology – the application of organisms/.

Biological systems/ processes in the manufacturing and.

Service industries.

Genetic engineering – is a tool in biotechnology and.

Involves heritable, directed alteration of an organism’s.

DNA.

Aim

Make a living cell perform a specific task in a predictable and

controllable way

Applications of Biotechnology

• Environmental – waste management• Medical – low amounts of high value product

A) diagnosis

B) therapyC) vaccineD) research toolsE) human genome

• Agricultural – large amount of low value product

More details in Chapter 12

Important Biotechonology Products - Medical

• Large scale production of penicillin.

• Monoclonal antibodies.

• Insulin was the first recombinant DNA derived product approved for human use.

• Human growth hormone was the second recombinant DNA derived product approved for human use.

Food and Drink Industry

• Uses of yeast

• Lactic acid fermentation

• Single cell proteins

• Bovine growth hormone – given to cows to inc milk production. More production than consumption. Gov. buys surplus

Service Industry-Enzyme Detergents

• Dirt comes in many forms and includes proteins, starches and lipids.

• Using detergents in water at high temperatures and with vigorous mixing, it is possible to remove most types of dirt.

• Cost of heating the water is high and lengthy mixing or beating will shorten the life of clothing and other materials.

Enzyme Detergents

• The use of enzymes allows lower temperatures to be employed and shorter periods of agitation are needed.

• At present only proteases and amylases are commonly used. Although a wide range of lipases is known, it is only very recently that lipases suitable for use in detergent preparations have been described.

Genetic Engineering

• Why genes?

• How can you change the genes of an individual?

• Examples of the use of this technology

Gene Vs Environment• DNA is the genetic material in all known forms of life. • DNA contains genes that give us many of our physical

characteristics. • The environment we are in also determines our traits.• One of the challenges of genetic engineering is to determine

how genes influence our traits and how to modify DNA to alter these traits.

• Genes affecting disorders such as alcoholism provide only a predisposition. Having the gene for alcoholism may make one more prone to alcoholism but does not guarantee that one will become alcoholic, nor does not having the gene mean one is immune.

Cells Used in Genetic Engineering• An important distinction in genetic engineering is between

germline and non-germline cells.• Germline cells are the eggs and sperm in humans. Non-germline

cells are all the other cells in the body - muscle cells, skin cells, liver, etc.

• If a genetic modification does not alter germline cells, it should not have any effect on the genetic makeup of future generations.

• If we were to introduce the gene for purple hair into mouse hair cells, the offspring would not have purple hair, but the parent would.

• If the gene for purple hair were introduced into the parental germline cells, then the children could carry the purple hair gene.

Dolly • No one thought that it would be possible to take a cell from an adult mammal

and use it to grow another, genetically identical clone. But that is how Dolly was created.

• She began life as a single cell taken from the udder of her mother. • The cell's nucleus was removed, transferred into an egg from which the DNA

had been removed, cultured and then implanted as an embryo into the womb of a surrogate sheep.

• Five months later, Dolly was born and is now a healthy lamb, a clone of her mother-and without a biological father.

• Dolly is very different from earlier sheep clones produced from cells taken from embryos. Cells at such an early stage are undifferentiated and it is not surprising that they can create all the different tissues of an adult organism as they divide.

• But cells from an adult were expected to be different-once an udder cell, always an udder cell. That the biological clock can be wound back and development run again as normal from the beginning is contrary to biological dogma.

• Now we know that we can clone an adult animal

Why Alter DNA?

• Since proteins don't last very long, they are poor targets for genetic engineering.

• Changes in DNA can be carried over generations, making it a good target for lasting changes.

• There are two basic types of modifications.A) addition - to add a function to a cell all you have to do is introduce a new gene that codes for the given function.B) deletion - deletion of function can be performed by either "knocking out" a gene or introducing an "antisense" gene to interfere with the cell's ability to express a given gene.

Tools of Genetic Engineering

• Restriction enzymes - used to cut DNA at specific points during production of rDNA.

• DNA ligase enzyme seals DNA fragments into the gap created by the restriction enzyme.

• Transfer of new genetic material back into the cell.

Transferring Foreign Genetic Material Into Plant Cells.

• Recombinant DNA uses bacterial plasmids and viruses to transport the new genes into the host cells.

• Microinjection involves injecting the genetic material into the recipient cells using a tiny glass needle. The injected genes then incorporate themselves among the host genes.

• Bioballistics use tiny slivers of metal that are coated with the genetic material. The slivers are "shot" into the host cells using a "gene gun". Once inside the cells, the new genes go to the nucleus and become incorporated with the host genes.

Cloning Steps

• Isolate and fragment of source DNA.

• Join source DNA to cloning vector.• Incorporate DNA into host.• Detect and purify desired clone.• Replicate clone to high numbers.

Restriction and Modification Enzymes

• Enzymes that cut DNA in specific places • Function: inactivate foreign DNA which

can derange metabolism • Breaks only palindrom sequences, i.E.

Those exhibiting two-fold symmetry mechanism

• Important in DNA research, i.E. Sequencing, hybridization companies purify and market restriction enzymes

OrganismEnzyme designation

Recognition sequence

Escherichia coli EcoRI G AATTC

Escherichia coli EcoRII CCAGG

Haemophilus hemolyticus

HhaI GCG C

Bacillus subtilus BsuRI G CCBrevibacterium albidum

BaII TGG CCA

Thermus aquaticus Taql T CGA

Examples

Cloned Cows Produce Human Antibodies

• Human Artificial Chromosomes (HACs) were introduced into cow cells that were then fused with bovine eggs from which the chromosomes had been removed.

• The embryos were then implanted in cows and allowed to develop into live calves.

• Even though the human genes were floating in their own chromosome, the researchers found the HAC was faithfully replicated by 80 per cent or more of the cow cells.

• When they examined immune cells from the animals, they found some had correctly assembled human antibody genes.

Genetic Engineering Plants

• Crops are engineered for several reasons.• Popular and successful engineering has allowed

for plants to possess tolerance to:-.- Herbicides, cheap pesticide used to kill weeds not harm crops.- Insects,- Viruses,- Diseases,

• Flavr-savr tomatoes no need to ripen artificially contain gene that inc shelf life.

Misuse of Biotechnology

• Companies – terminator genes

• Biological warfare

Human Genome Project

• Begun in 1990, project goals were to:-.

- Identify all the approximately 30,000 genes in human DNA,

Determine the sequences of the 3 billion chemical base pairs that make up human DNA,

Store this information in databases,

Improve tools for data analysis,

Transfer related technologies to the private sector, and.

Address the ethical, legal, and social issues (ELSI) that may arise from the project.

Human Genome Project

• A working draft of the entire human genome sequence was announced in June 2000,

• Analyses published in February 2001.

• An important feature of this project is the federal government's long-standing dedication to the transfer of technology to the private sector. By licensing technologies to private companies and awarding grants for innovative research, the project is catalyzing the multibillion-dollar U.S. Biotechnology industry and fostering the development of new medical applications.

Diagnosing and Predicting Disease and Disease Susceptibility

• All diseases have a genetic component, whether inherited or resulting from the body's response to environmental stresses like viruses or toxins.

• The ultimate goal is to use this information to develop new ways to treat, cure, or even prevent the thousands of diseases that afflict humankind.

• In the meantime, biotechnology companies are racing ahead with commercialization by designing diagnostic tests to detect errant genes in people suspected of having particular diseases or at risk for developing them.

• The potential for using genes themselves to treat disease--known as gene therapy--is the most exciting application of DNA science.

• Genes - through the proteins they encode - determine how efficiently we process foods, how effectively we detoxify poisons, and how vigorously we respond to infections.

• More than 4,000 diseases are thought to stem from mutated genes inherited from one's mother and/or father.

• Common disorders such as heart disease and most cancers arise from a complex interplay among multiple genes and between genes and factors in the environment

Mutations• A hereditary mutation is a mistake that is present in the DNA of

virtually all body cells. Hereditary mutations are also called germline mutations because the gene change exists in the reproductive cells (germ cells) and can be passed from generation to generation, from parent to newborn. Moreover, the mutation is copied every time body cells divide.

• Acquired mutations, also known as somatic mutations, are changes in DNA that develop throughout a person's life. In contrast to hereditary mutations, somatic mutations arise in the DNA of individual cells; The genetic errors are passed only to direct descendants of those cells. Mutations are often the result of errors that crop up during cell division, when the cell is making a copy of itself and dividing into two. Acquired mutations can also be the byproducts of environmental stresses such as radiation or toxins.

Mutations

• Mutations occur all the time in every cell in the body.

• Each cell, however, has the remarkable ability to recognize mistakes and fix them before it passes them along to its descendants.

• But a cell's DNA repair mechanisms can fail, or be overwhelmed, or become less efficient with age. Over time, mistakes can accumulate.

Genetic Testing• Genetic tests can be used to look for possible predisposition to

disease as well as to confirm a suspected mutation. • Newborn screening - most widespread type of genetic testing some

tests look for abnormal arrangements of the chemical bases in the gene itself, while other tests detect inborn errors of metabolism (for example, phenylketonuria) by verifying the absence of a protein that the cell needs to function normally.

• Carrier testing can be used to help couples to learn if they carry - and thus risk passing to their children - a recessive allele for inherited disorders such as cystic fibrosis, sickle-cell anemia, or Tay-Sachs disease (a lethal disorder of lipid metabolism).

• Prenatal diagnosis - also are widely available for the of conditions such as down syndrome, beta-Thalassaemia.

Genetic Tests• Doctors make use of genetic tests to identify telltale DNA

changes in cancer or precancer cells.• Such tests can be helpful in several areas: early detection

(familial adenomatous polyposis genes prompt close surveillance for colon cancer); Diagnosis (different types of leukemia can be distinguished); Prognosis (the product of a mutated p53 tumor-suppressor gene flags cancers that are likely to grow aggressively); And treatment (antibodies block a gene product that promotes the growth of breast cancer).

• Much of the current excitement in gene testing, however, centers on predictive gene testing: tests that identify people who are at risk of getting a disease, before any symptoms appear. Tests are already available in research programs for some two dozen such diseases, and as more disease genes are discovered, more gene tests can be expected.

Gene Therapy• A novel approach to treat, cure, or ultimately prevent

disease by changing the expression of a person's genes. • Current gene therapy is primarily experimental, with most

human clinical trials only in the research stages. • Gene therapy can be targeted to somatic (body) or germ

(egg and sperm) cells. • Many people falsely assume that germline gene therapy

already is being done with regularity. • News reports of parents selecting a genetically tested egg

for implantation or choosing the sex of their unborn child may lead the public to think that gene therapy is occuring. Actually, in these cases, genetic information is being used for selection. No cells are altered or changed.

First Actual Case of Gene Therapy • Deficiency of the protein adenosine deaminase "ADA“,

involved in maturation of T and B cells. • Causes collapse of the immune system.• Remove stem cells or WBC’s by taking blood samples.• Culture cells & genetically engineer with recombinant

DNA containing good ADA gene using a viral vector.• Reintroduce cultured cells into host (transfusion.• New stem cells and /or WBC’s make ADA and immune

system recovers.• This procedure to recover function of ADA only worked

because blood cells are easy to remove and to reintroduce.

Gene Therapy Trails

• Children with severe combined immunodeficiency syndrome (SCD) underwent ex vivo gene therapy.

• Gene therapy trials include treatment of hypercholesterolemia where liver cells lack a receptor for removing cholesterol from blood.

• Cystic fibrosis patients lack a gene for trans-membrane chloride ion carriers; patients die from respiratory tract infections. Liposomes, microscopic vesicles that form when lipoproteins are in solution, are coated with healthy cystic fibrosis genes and sprayed into a patient's nostrils. This method is in vivo method.

PCR• Usually only tiny amounts of DNA can be isolated from

individual living organisms, too little for engineering purposes.

• DNA can be amplified by "cloning“, i.e. adding recombinant DNA to bacteria and grow up many kg of bacteria.

• A more recent technique is the polymerase chain reaction or PCR.

• Millions of copies of a DNA fragment can be made in a few hours entirely in vitro.

• Uses DNA polymerase, the enzyme normally used by bacterial cells to make copies of DNA in the cell cycle.

• Can amplify tiny amounts of DNA from fossils, embryonic cells, tissue or semen samples from crime victims.

• "Jurassic park."

• Genes exist in different forms called alleles.• When cellular DNA is cut with restriction enzymes they form

restriction fragments of different sizes depending on the alleles present.

• Different sized fragments when separated on a gel show "restriction fragment length polymorphisms" (RFLPS).

• Since each individual has different alleles for their genes, each individual will show a characteristic "DNA fingerprint" .

• Hasn’t always been acceptable as a form of proof of guilt in criminal investigations but is very useful in eliminating suspects.

• Has been used to identify rapists and murderers and in some cases to release innocent persons from prison.

DNA Fingerprinting

Mitochondrial DNA Typing• When nuclear DNA in small amounts.• Hundreds of copies of mtDNA for every copy of nuclear DNA.• Maternally inherited.• Used to identify remains of the Russian Royal family.• Nine skeletons had been found in a shallow grave in Ekaterinburg,

Russia, in July 1991. • Mitochondrial DNA was obtained and sequenced from the remains

by a team from Moscow, Cambridge and the UK FSS. • The skeleton thought to be that of the Tsarina did indeed have the

same sequence as the three thought to be her children. • Furthermore, the sequence also matched that from the Duke of

Edinburgh, who is related through the female line to the Tsarina and who would therefore be expected to have the same mitochondrial DNA

Ethical Questions Concerning Genetic Engineering on Humans

 • When and on whom should genetic testing be done?• Who should have access to results of genetic testing?• Should people be denied employment or insurance because

they have genetic profiles deemed "defective"?• With spiraling medical costs, should carriers of genetic

disorders be denied the right to have children? Forced to pay high insurance premiums?

• Do humans have the right to produce and patent new transgenic species?

• What about medical researchers patenting genetic material from cells derived from your body with or without your consent?