GENETIC ENGINEERING FEBRUARY 7 2013 MRS HAUGHTON CAPE

GENETIC ENGINEERING FEBRUARY 7, 2013 MRS. HAUGHTON CAPE BIOLOGY

• Genetic engineering, also known as recombinant DNA technology, means altering the genes in a living organism to produce a Genetically Modified Organism (GMO) with a new genotype.

• 1. 2. 3. 4. Various kinds of genetic modification are possible: inserting a foreign gene from one species into another forming a transgenic organism altering an existing gene so that its product is changed changing gene expression so that it is translated more often or not at all.

TECHNIQUES OF GENETIC ENGINEERING

• Genetic engineering is a very young discipline, and is only possible due to the development of techniques from the 1960 s onwards. • These techniques have been made possible from our greater understanding of DNA and how it functions following the discovery of its structure by Watson and Crick in 1953.

• Although the final goal of genetic engineering is usually the expression of a gene in a host, in fact most of the techniques and time in genetic engineering are spent isolating a gene and then cloning it.

TECHNIQUE PURPOSE Restriction enzymes To cut DNA at specific points, making small fragments DNA ligase To join DNA fragments together Vectors To carry DNA into cells and ensure replication Plasmids Common kind of vector Genetic markers To identify cells that have been transformed

TECHNIQUE PURPOSE Polymerase chain reaction (PCR) To amplify very small pieces of DNA c. DNA To make a DNA copy of RNA DNA probes Gene synthesis To identify and label a piece of DNA containing a certain sequence To make a gene from scratch Electrophoresis To separate fragments of DNA Sequencing To read the base sequence of a length of DNA

RECOMBINANT DNA TECHNOLOGY

• Genetic engineering in bacteria can be broken down into six stages:

1 • Recombinant technology begins with the isolation of a gene of interest. The gene is then inserted into a vector and cloned.

2 • A vector is a piece of DNA that is capable of independent growth; commonly used vectors are bacterial plasmids and viral phages. • The gene of interest (foreign DNA) is integrated into the plasmid or phage, and this is referred to as recombinant DNA.

3 • Before introducing the vector containing the foreign DNA into host cells to express the protein, it must be cloned. Cloning is necessary to produce numerous copies of the DNA since the initial supply is inadequate to insert into host cells.

5 • Once the vector is isolated in large quantities, it can be introduced into the desired host cells such as mammalian, yeast, or special bacterial cells. • The host cells will then synthesize the foreign protein from the recombinant DNA.

6 • When the cells are grown in vast quantities, the foreign or recombinant protein can be isolated and purified in large amounts.

Other uses for recombinant DNA • Recombinant DNA technology is not only an important tool in scientific research, but it has also impacted the diagnosis and treatment of diseases and genetic disorders in many areas of medicine. • It has enabled many advances, including:

Isolation of large quantities of protein • In addition to the folliclestimulating hormone, insulin, growth hormone and other proteins are now available as recombinant products.

Identification of mutations • People may be tested for the presence of mutated proteins that may be associated with breast cancer, retino-blastoma, and neurofibromatosis.

Diagnosis of affected and carrier states for hereditary diseases • Tests exist to determine if people are carriers of the cystic fibrosis gene, the Huntington’s disease gene, the Tay-Sachs disease gene, or the Duchenne muscular dystrophy gene.

Transferring of genes from one organism to another • People suffering from cystic fibrosis, rheumatoid arthritis, vascular disease, and certain cancers may now benefit from the progress made in gene therapy.

Mapping of human genes on chromosomes • Scientists are able to link mutations and disease states to specific sites on chromosomes.

RESTRICTION ENDONUCLEASES

• Restriction enzymes are DNA-cutting enzymes found in bacteria (and harvested from them for use). • Because they cut within the molecule, they are often called restriction endonucleases.

• In order to be able to sequence DNA, it is first necessary to cut it into smaller fragments. • Many DNA-digesting enzymes (like those in your pancreatic fluid) can do this, but most of them are not used for sequence work because they cut each molecule randomly. • This produces a heterogeneous collection of fragments of varying sizes.

• What is needed is a way to cleave the DNA molecule at a few precisely-located sites so that a small set of homogeneous fragments are produced. • The tools for this are the restriction endonucleases. The rarer the site it recognizes, the smaller the number of pieces produced by a given restriction endonuclease.

• A restriction enzyme recognizes and cuts DNA only at a particular sequence of nucleotides. • For example, the bacterium Hemophilus aegypticus produces an enzyme named Hae. III that cuts DNA wherever it encounters the sequence 5'GGCC 3' 3'CCGG 5‘. • The cut is made between the adjacent G and C.

• This particular sequence occurs at 11 places in the circular DNA molecule of the virus φX 174. • Thus treatment of this DNA with the enzyme produces 11 fragments, each with a precise length and nucleotide sequence. • These fragments can be separated from one another and the sequence of each determined.

• Hae. III and Alu. I cut straight across the double helix producing "blunt" ends. However, many restriction enzymes cut in an offset fashion. • The ends of the cut have an overhanging piece of single-stranded DNA. These are called "sticky ends" because they are able to form base pairs with any DNA molecule that contains the complementary sticky end. • Any other source of DNA treated with the same enzyme will produce such molecules.

• Mixed together, these molecules can join with each other by the base pairing between their sticky ends. The union can be made permanent by another enzyme, DNA ligase, that forms covalent bonds along the backbone of each strand. • The result is a molecule of recombinant DNA (r. DNA).

• Recombinant DNA molecules have revolutionized the study of genetics and laid the foundation for much of the biotechnology industry. • The availability of human insulin (for diabetics), human factor VIII (for males with hemophilia A), and other proteins used in human hormone therapy all were made possible by recombinant DNA.

• Restriction enzyme 1 • http: //www. youtube. com/watch? v=a. A 5 fy. W Jh 5 S 0 • http: //www. youtube. com/watch? v=-s. I 5 vyc. D 2 g

Applications of Recombinant DNA Technology

Human Applications • Treatment of genetic disorders. Medical scientists now know of about 3, 000 disorders that arise because of errors in an individual's DNA. • Conditions such as sickle-cell anemia, Tay. Sachs disease, Duchenne muscular dystrophy, Huntington's chorea, cystic fibrosis, and Lesch. Nyhan syndrome are the result of the loss, mistaken insertion, or change of a single nitrogen base in a DNA molecule. •

• Genetic engineering makes it possible for scientists to provide individuals who lack a certain gene with correct copies of that gene. • For instance, in 1990 a girl with a disease caused by a defect in a single gene was treated in the following fashion. Some of her blood was taken, and the missing gene was copied and inserted into her own white blood cells, then the blood was returned to her body. • If—and when—that correct gene begins to function, the genetic disorder may be cured. This type of procedure is known as human gene therapy (HGT)

Agricultural Applications • It is now possible to produce plants that will survive freezing temperatures, take longer to ripen, convert atmospheric nitrogen to a form they can use, manufacture their own resistance to pests, and so on. • By 1988 scientists had tested more than two dozen kinds of plants engineered to have special properties such as these.

• Domestic animals have been genetically "engineered" in an inexact way through breeding programs to create more meaty animals, etc. , but with genetic engineering, these desirable traits could be guaranteed for each new generation of animal.

GENE THERAPY

• Gene therapy is the insertion, alteration, or removal of genes within an individual's cells and biological tissues to treat disease. • It is a technique for correcting defective genes that are responsible for disease development.

• The most common form of gene therapy involves the insertion of functional genes into an unspecified genomic location in order to replace a mutated gene, but other forms involve directly correcting the mutation or modifying normal gene that enables a viral infection. • Although the technology is still in its infancy, it has been used with some success.

Types of gene therapy • Gene therapy may be classified into the two following types: • Germ line gene therapy • Somatic gene therapy

Germ line gene therapy • In the case of germ line gene therapy, germ cells, i. e. , sperm or eggs, are modified by the introduction of functional genes, which are integrated into their genomes. • Therefore, the change due to therapy would be heritable and would be passed on to later generations.

Germ line gene therapy • This new approach, theoretically, should be highly effective in counteracting genetic disorders and hereditary diseases. However, many jurisdictions prohibit this for application in human beings, at least for the present, for a variety of technical and ethical reasons.

Somatic gene therapy • In the case of somatic gene therapy, therapeutic genes are transferred into the somatic cells of a patient. • Any modifications and effects will be restricted to the individual patient only, and will not be inherited by the patient's offspring or later generations.

ETHICS

DESIGNER BABIES

Vectors in gene therapy

Viruses • All viruses bind to their hosts and introduce their genetic material into the host cell as part of their replication cycle. • This genetic material contains basic 'instructions' of how to produce more copies of these viruses, hijacking the body's normal production machinery to serve the needs of the virus.

Retroviruses • The genetic material in retroviruses is in the form of RNA molecules, while the genetic material of their hosts is in the form of DNA. • When a retrovirus infects a host cell, it will introduce its RNA together with some enzymes, namely reverse transcriptase and integrase, into the cell.

Retroviruses • This RNA molecule from the retrovirus must produce a DNA copy from its RNA molecule before it can be integrated into the genetic material of the host cell.

Adenoviruses • Adenoviruses are viruses that carry their genetic material in the form of double-stranded DNA. • They cause respiratory, intestinal, and eye infections in humans (especially the common cold). • When these viruses infect a host cell, they introduce their DNA molecule into the host. • The genetic material of the adenoviruses is not incorporated (transient) into the host cell's genetic material.

Adenoviruses • The DNA molecule is left free in the nucleus of the host cell, and the instructions in this extra DNA molecule are transcribed just like any other gene. • The only difference is that these extra genes are not replicated when the cell is about to undergo cell division so the descendants of that cell will not have the extra gene. • As a result, treatment with the adenovirus will require re-administration in a growing cell population.

Injection of Naked DNA • This is the simplest method of non-viral transfection. • Cellular uptake of naked DNA is generally inefficient. • Research efforts focusing on improving the efficiency of naked DNA uptake have yielded several novel methods, such as the use of a "gene gun", which shoots DNA coated gold particles into the cell using high pressure gas.

Physical Methods to Enhance Delivery

Electroporation • Electorporation is a method that uses short pulses of high voltage to carry DNA across the cell membrane. • This shock is thought to cause temporary formation of pores in the cell membrane, allowing DNA molecules to pass through. • Electroporation is generally efficient and works across a broad range of cell types. However, a high rate of cell death following electroporation has limited its use, including clinical applications.

Gene Gun • The use of particle bombardment, or the gene gun, is another physical method of DNA transfection. • In this technique, DNA is coated with gold particles and loaded into a device which generates a force to achieve penetration of DNA/gold into the cells.

Sonoporation • Sonoporation uses ultrasonic frequencies to deliver DNA into cells. The process of acoustic cavitation is thought to disrupt the cell membrane and allow DNA to move into cells.

Magnetofection • In a method termed magnetofection, DNA is complexed to a magnetic particles, and a magnet is placed underneath the tissue culture dish to bring DNA complexes into contact with a cell monolayer.

Problems and ethics • Short-lived nature of gene therapy – Before gene therapy can become a permanent cure for any condition, therapeutic DNA introduced into target cells must remain functional and the cells containing therapeutic DNA must be longlived and stable. • Immune response – Anytime a foreign object is introduced into human tissues, the immune system has evolved to attack the invader.

• Problems with viral vectors – Viruses, the carrier of choice in most gene therapy studies, present a variety of potential problems to the patient —toxicity, immune and inflammatory responses, and gene control and targeting issues. In addition, there is always the fear that the viral vector, once inside the patient, may recover its ability to cause disease.

• Multigene disorders – Conditions or disorders that arise from mutations in a single gene are the best candidates for gene therapy. • Chance of inducing a tumor (insertional mutagenesis) - If the DNA is integrated in the wrong place in the genome, for example in a tumor suppressor gene, it could induce a tumor. • Deaths may occur.

• What are the ethical issues surrounding gene therapy? • Because gene therapy involves making changes to the body’s set of basic instructions, it raises many unique ethical concerns. The ethical questions surrounding gene therapy include: • How can “good” and “bad” uses of gene therapy be distinguished? • Who decides which traits are normal and which constitute a disability or disorder? • Will the high costs of gene therapy make it available only to the wealthy? • Could the widespread use of gene therapy make society less accepting of people who are different? • Should people be allowed to use gene therapy to enhance basic human traits such as height, intelligence, or athletic ability?

PROS OF GENETIC ENGINEERING

• Crops like potato, tomato, soybean and rice are currently being genetically engineered to obtain new strains with better nutritional qualities and increased yield. • The genetically engineered crops are expected to have a capacity to grow on lands that are presently not suitable for cultivation. • The manipulation of the genes in crops is expected to improve their nutritional value as also their rate of growth. Biotechnology, the science of genetically engineering foods, can be used to impart a better taste to certain foods.

• Engineered seeds are resistant to pests and can survive in a relatively harsh climatic conditions. • The recently identified plant gene known as At. DBF 2, when inserted in tomato and tobacco cells is seen to increase their endurance to harsh soil and climatic conditions. • Biotechnology can be used to slow down the process of food spoilage. It can thus result in fruits and vegetables having a greater shelf life.

• Genetic engineering in food can be used to produce totally new substances such as proteins and other food nutrients. The genetic modification of foods can be used to increase their medicinal value, thus making available homegrown edible vaccines.

• Genetic engineering has a great potential of succeeding in case of human beings. This specialized branch of genetic engineering, which is known as human genetic engineering is the science of modifying the genotypes of human beings before birth. The process can be used to manipulate certain traits in an individual.

• Positive genetic engineering deals with enhancing the positive traits in an individual like increasing longevity or human capacity while negative genetic engineering deals with the suppression of the negative traits in human beings like certain genetic diseases. Genetic engineering can be used to obtain a permanent cure for certain dreaded diseases.

• If the genes responsible for the exceptional qualities in some individuals can be discovered, these genes can be artificially introduced into genotypes of other human beings. Genetic engineering in human beings can be used to change the DNA of individuals to bring about desirable structural and functional changes in them.

CONS OF GENETIC ENGINEERING

• Genetic engineering in food involves the contamination of genes in crops. Genetically engineered crops may supersede the natural weeds; they may prove harmful for the natural plants. Undesirable genetic mutations can lead to allergies in crops. Critics believe that genetic engineering in foodstuffs can rather hamper the nutritional value while enhancing their taste and appearance.

• Horizontal gene transfer can give rise to new pathogens. While increasing the immunity to diseases in plants, the resistance genes may get transferred to the harmful pathogens.

• Gene therapy in human beings can manifest certain side effects. While treating one defect, therapy may lead to another. As one cell is responsible for many characteristics, the isolation of the cells responsible for a single trait is indeed difficult.

• Genetic engineering can hamper the diversity in human beings. Cloning can be detrimental to individuality. Moreover, such processes may not be affordable for the masses, thus making gene therapy, an impossibility for the common man.

• Genetic engineering may work wonders but it is after all a process of manipulating the nature. It is altering something that is not an original human creation. Modifying something that one has not created is always challenging.