Recombinant DNA Technology Chinese Dragon The Chinese Dragon

Recombinant DNA Technology

Chinese Dragon 龙

The Chinese Dragon n The dragon is an imaginary creature which has deer's antlers, camel's head, rabbit's eyes, snake's neck, carp's scales, eagle's claws, tiger's paws, and ox's ears.

Recombinant DNA n Recombinant DNA (r. DNA) is a form of artificial DNA that is created by combining two or more sequences that would not normally occur together through the process of gene splicing. n Recombinant DNA technology is technology which allows DNA to be produced via artificial means. The procedure has been used to change DNA in living organisms and may have even more practical uses in the future.

Recombinant DNA Technology 1. The basic concepts for recombinant DNA technology 2. The basic procedures of recombinant DNA technology 3. Application of recombinant DNA technology

n In the early 1970 s, technologies for the laboratory manipulation of nucleic acids emerged. n The products of these innovations, recombinant DNA molecules, opened many new avenues of investigation in the fields of molecular biology and genetics.

Concept of Recombinant DNA n r. DNA creates a new combination of genetic material The human gene for insulin was placed in bacteria The bacteria are recombinant organisms and produce insulin in large quantities for diabetics A genetically engineered form of insulin became available in 1986

n Genetic engineering is the application of this technology to the manipulation of genes. These advances were made possible by methods for amplification of any particular DNA segment. n Once amplification was possible, the cloning of virtually any DNA sequence became feasible.

Six Steps of Recombinant DNA 1. Isolating (vector and target gene) 2. Cutting (Cleavage) 3. Joining (Ligation) 4. Transforming 5. Cloning 6. Selecting (Screening)

Recombinant DNA Technology 1. The basic concepts for recombinant DNA technology 2. The basic procedures of recombinant DNA technology 3. Application of recombinant DNA technology

3. Ligation of DNA fragments. A recombinant DNA molecule is usually formed by cleaving the DNA of interest to yield insert DNA and then ligating the insert DNA to vector DNA (recombinant DNA). DNA fragments are typically joined using DNA ligase. What type of molecule is DNA ligase…. . ?

5. Replication and expression of recombinant DNA in host cells. Cloning vectors allow insert DNA to be replicated and, in some cases, expressed in a host cell. The ability to clone and express DNA efficiently depends on the choice of appropriate vectors and hosts.

6. Identification of host cells that contain recombinant DNA of interest. Vectors usually contain genetic markers that allow the selection of host cells that have taken up foreign DNA. The identification of a particular DNA fragment usually involves an additional step—screening a large number of recombinant DNA clones. This is almost always the most difficult step.

DNA cloning in a plasmid vector permits amplification of a DNA fragment.

Applications of Recombinant DNA Technology

1. Analysis of Gene Structure and Expression 2. Pharmaceutical Products 3. Drugs Vaccines Genetically modified organisms Transgenic plants Transgenic animal 4. Application in medicine 5. (GMO)

Analysis of Gene Structure and Expression n Using specialized recombinant DNA techniques, researchers have mapped the entire genomic sequence of humans and many key experimental organisms. This enormous volume of data, which is growing at a rapid pace, has been stored and organized in two primary data banks: n Gen. Bank at the National Institutes of Health, Bethesda, Maryland n EMBL Sequence Data Base at the European Molecular Biology Laboratory in Heidelberg, Germany.

Pharmaceutical Products n n Some pharmaceutical applications of DNA technology: n Large-scale production of human hormones and other proteins with therapeutic uses n Production of safer vaccines A number of therapeutic gene products —insulin, the interleukins, interferons, growth hormones, erythropoietin, and coagulation factor VIII— are now produced commercially from cloned genes

Insulin Hormone required to properly process sugars and fats Treat diabetes Now easily produced by bacteria Growth hormone deficiency Faulty pituitary and regulation Had to rely on cadaver source Now easily produced by bacteria

Not always used for good. . . High doses of HGH can cause permanent side effects Bone thickening Enlarged organs Diabetes High blood pressure Reduced fertility

Genetically modified organisms (GMO) Plants with genetically desirable traits herbicide or pesticide resistant corn & soybean Decreases chemical insecticide use Increases production “Golden rice” with beta-carotene Required to make vitamin A, which in deficiency causes blindness

Genetic Engineering of Plants n Plants have been bred for millennia to enhance certain desirable characteristics in important food crops. n Transgenic plants have developed significantly over the past 20 years as technology has evolved.

The luciferase gene from a firefly is transformed into tobacco plant using the Ti plasmid. Watering the plant with a solution of luciferin (the substrate for firefly luciferase) results in the generation of light by all plant tissues.

Insect-resistant tomato plants The plant on the left contains a gene that encodes a bacterial protein that is toxic to certain insects that feed on tomato plants. The plant on the right is a wildtype plant. Only the plant on the left is able to grow when exposed to the insects.

Transgenic animals Green fluorescence Red fluorescence

Transgenic animals

A transgenic mouse Mouse on right is normal; mouse on left is transgenic animal expressing rat growth hormone

Farm Animals and “Pharm” Animals n n Trangenic plants and animals have genes from other organisms. These transgenic sheep carry a gene for a human blood protein – This protein may help in the treatment of cystic fibrosis

Other benefits of GMOs n Disease resistance n n n Cold tolerance n n There are many viruses, fungi, bacteria that cause plant diseases “Super-shrimp” Antifreeze gene from cold water fish introduced to tobacco and potato plants Drought tolerance & Salinity tolerance n As populations expand, potential to grow crops in otherwise inhospitable environments

Where in the world?

Downsides? ? ? n Introduce allergens? n Pass trans-genes to wild populations? n Pollinator transfer R&D is costly Patents to insure profits Patent infringements Lawsuits potential for capitalism to overshadow humanitarian efforts

Application in medicine n Human Gene Therapy n Diagnosis of genetic disorders n Forensic Evidence

Human Gene Therapy n Human gene therapy seeks to repair the damage caused by a genetic deficiency through introduction of a functional version of the defective gene. To achieve this end, a cloned variant of the gene must be incorporated into the organism in such a manner that it is expressed only at the proper time and only in appropriate cell types. At this time, these conditions impose serious technical and clinical difficulties.

n Gene therapy is the alteration of an afflicted individual’s genes n Gene therapy holds great potential for treating disorders traceable to a single defective gene n Vectors are used for delivery of genes into cells Gene therapy raises ethical questions, such as whether human germ-line cells should be treated to correct the defect in future generations n

n Many gene therapies have received approval from the National Institutes of Health for trials in human patients, including the introduction of gene constructs into patients. Among these are constructs designed to cure ADA- SCID (severe combined immunodeficiency due to adenosine deaminase [ADA] deficiency), neuroblastoma, or cystic fibrosis, or to treat cancer through expression of the E 1 A and p 53 tumor suppressor genes.

Cloned gene Insert RNA version of normal allele into retrovirus. Viral RNA Retrovirus capsid Let retrovirus infect bone marrow cells that have been removed from the patient and cultured. Somatic cells Only! Viral DNA carrying the normal allele inserts into chromosome. Bone marrow cell from patient Inject engineered cells into patient. Bone marrow Not for reproductive cells !!

However, there are some challenging issues that need to be considered: 1. In mammalian cells, m. RNA is processed before it is translated into a protein: Introns are cut out and exons are spliced together Bacteria can not process m. RNA

2. Post-translational modifications Enzymatic modifications of protein molecules after they are synthesized in cells Post-translational modifications include: Disulfide bond formation (catalyzed by disulfide isomerases) and protein folding Glycosylation (addition of sugar molecules to protein backbone, catalyzed by glycosyl transferases) Proteolysis (clipping of protein molecule, e. g. , processing of proinsulin to insulin) Sulfation, phosphorylation (addition of sulfate, phosphate groups)

3. Recombinant proteins are particularly susceptible to proteolytic degradation in bacteria 4. Recombinant protein may accumulate in bacteria as refractile inclusion bodies

So how can these problems be tackled? Problem: 1. m. RNA processing in mammalian cells but not in bacteria Solution: Synthesize chemically gene containing only exons and insert that into vector; or, Make c. DNA by reverse-transcription of processed m. RNA (using the enzyme reverse transcriptase)

Problem: 2. Bacteria cannot perform post-translational modifications Solution: This is a tough one! Only proteins that do not undergo extensive post-translational processing can be synthesized in bacteria

Problem: 3. Recombinant proteins particularly susceptible to proteolysis Solution: Design fusion protein consisting of an endogenous bacterial protein connected to the recombinant protein through a specific amino acid sequence. Fusion protein is then specifically cleaved at the fusion site

The Hope

Summary 1. Recombinant DNA technology builds on a few basic techniques: isolation of DNA, cleavage of DNA at particular sequences, ligation of DNA fragments, introduction of DNA into host cells, replication and expression of DNA, and identification of host cells that contain recombinants.

2. DNA Fragments generated by restriction endonucleases can be ligated into a wide range of cloning vectors, including: plasmids, bacteriophage, viruses, or artificial chromosomes.

3. Cells containing recombinant DNA molecules can be selected, often by the activity of a marker gene. Cells containing the desired recombinant are identified by screening.

4. The product of a gene that has been incorporated into an appropriate expression vector can be generated in prokaryotic or eukaryotic cells. Foreign genes can also be stably incorporated into the genomes of animals and plants.

5. Recombinant DNA methods allow the production of proteins for therapeutic use and the identification of individuals with genetic defects.

REVIEW QUESTIONS Choose the ONE BEST answer or completion. Plasmids used as cloning vectors A. are circular molecules. B. have an origin of replication. C. carry antibiotic resistance genes. D. have unique restriction endonuclease cutting sites. E. all of the above.