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Biotechnology is broadly defined as technologies that involve the use of living organisms, or their products, to benefit humans It is not a new topic It began about 12,000 years ago when humans began to domesticate animal and plants for the production of food Since the 1970s, molecular genetics has provided new, improved ways to make use of organisms to benefit humans Genetically modified organisms (GMOs) have received genetic material via recombinant DNA technology An organism that has integrated recombinant DNA into its genome is called transgenic Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display USES OF MICROORGANISMS IN BIOTECHNOLGY Microorganisms are used to benefit humans in various ways Molecular genetic tools are very important in influencing and improving our use of microorganisms Overall, the use of recombinant microorganisms is an area of great research interest and potential However, there are problems such as safety concerns and negative public perception Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Many Important Medicines Are Produced by Recombinant Microorganisms Insulin Insulin regulates several physiological processes, particularly the uptake of glucose into fat and muscle cells Persons with insulin-dependent diabetes have a defect in their b cells Therefore, they cannot synthesize enough insulin Sources of insulin included It is produced by the b cells of the pancreas Cows Human cadavers! But now, patients can use insulin made by recombinant bacteria – expression vector – eukaryotic gene expressed in a bacteria Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display GENETICALLY MODIFIED ANIMALS The production of transgenic animals is a relatively new, exciting area of biotechnology It holds great promise for innovations in biotechnology Of course, this is predicated on public acceptance! Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Genes can be introduced from different species. Here, different fluorescent proteins from jellyfish are expressed in zebrafish. Glo fish Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Transgenic Livestock Transgenic species of livestock are being developed May include production of medicines in the milk of these animals molecular pharming Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display REPRODUCTIVE CLONING Reproductive cloning refers to methods that produce two or more genetically identical individuals Cloning is an easier undertaking in plants Identical twins are genetic clones from one fertilized egg Plants can be cloned from somatic cells For several decades scientists believed that mammalian somatic cells were unsuitable for cloning But in 1997, Ian Wilmut and his colleagues at the Roslin Institute created Dolly! Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Dolly and her lamb Bonnie Protocol for the successful cloning of sheep Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Protocol for the successful cloning of sheep Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Evidence suggested that Dolly may be “genetically older” than her actual age would have indicated At 3 years old, the length of the telomeres in her somatic cells were consistent with a sheep that is 9 or 10 years old The sheep that donated the somatic cell that produced Dolly was 6 years old 6-year old Dolly was euthanized after an examination showed progressive lung disease Her death raised concerns that the techniques used to produce Dolly could have caused premature aging As much as 4% of genes were not expressed normally Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display In recent years, cloning from somatic cells has been achieved in several mammalian species Sheep, cows, mice, goats and pigs Unlike the case with Dolly, telomeres in cloned mice and cattle appear to be the correct length! However, other studies have shown other genetic flaws With regard to livestock, farmers can use somatic cells from their best individuals to create genetically homogeneous herds This may be advantageous with regard to agricultural yield However, such a herd may be more susceptible to rare diseases Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Cloned cats and dogs Cloned cows Cloned horses People have become greatly concerned with the possibility of human cloning To some, it is morally wrong and threatens the basic fabric of parenthood and family To others, it offers a new avenue of reproduction For infertile couples who might want a genetically related child In the public sector, the sentiment toward human cloning has been generally negative Indeed, many countries have issued an all-out ban While others permit limited research in the area In the future our society will have to wrestle with the legal and ethical aspects of cloning Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Stem Cells Stem cells supply the cells that construct our bodies from a fertilized egg In the adult, stem cells also replenish damaged cells Stem cells have two common characteristics 1. They have the capacity to divide 2. They have the capacity to differentiate into one or more specialized cell types totipotent cells, like fertilized eggs can give rise to all cell types pluripotent cells can differentiate into almost every cell, but can’t give rise to an entire, intact individual multipotent cells can differentiate into several cell types unipotent cells can only differentiate into one cell type Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display When stem cell divides, one may remain undifferentiated, while the other can differentiate into a specialized cell type Thus the population of stem cells remains constant Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display In mammals, stem cells are commonly categorized based on the developmental stage and the ability to differentiate Can produce all cell types in an adult organism Can give rise to an entire organism Found in the early mammalian embryo Can produce almost all cell types in an adult organism However, a single cell has lost the ability to produce an entire organism Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Can differentiate into several cells types For example, hematopoietic stem cells (blood cells) of the bone marrow Can only differentiate into a single cell type For example, primordial germ cells in the testis sperm, only Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Stem Cells Adult stem cells are rare 1 cell in 10,000 in the bone marrow is a stem cell Embryonic Stem cells (ES) and Embryonic Germ cells (EG) can be grown in the laboratory Are easily identified Provide greatest potential for transplantation therapy Most ES cells are derived from unused embryos from in vitro fertilization Most EG cells are derived from aborted fetuses This creates an ethical dilemma in using these cells Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Genetically Modified Plants Selective breeding has been used for centuries This has produced plants with desirable characteristics larger, disease resistance, high-quality food Genetically engineered crops have been used since mid-1990s In 2009, roughly 25% of all crops were transgenic More than 100 million hectares planted with transgenics Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Agrobacterium tumefaciens Can Be Used to Make Transgenic Plants The production of transgenic plants is somewhat easier than transgenic animals Certain plant cells are totipotent An entire organism can be regenerated from a somatic cell Agrobacterium tumefaciens is a bacterium that naturally infects plants causing crown gall tumor Ti plasmid (Tumor-inducing) carried by bacterium Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Transferred DNA Tumor-inducing plasmid Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The A. tumefaciens T DNA can be used as a vector to introduce cloned genes into plants First, the Ti plasmid needs to be modified The genes that cause tumors are deleted Selectable marker genes are inserted into the T DNA R is commonly used Kan Unique restriction sites are added for the convenient insertion of any gene Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display T DNA integrates into the genome Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display A. tumefaciens does not infect all plant species Fortunately, other methods are available Biolistic gene transfer (i.e., biological ballistics) Microinjection The second most common way to produce transgenic plants A “DNA gun” is used to shoot DNA-coated microprojectiles into the cells Microscopic-sized needles are used to inject DNA into the cells Electroporation An electric current is used to create transient pores in the plasma membrane through which DNA can enter Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Potato plants transformed with a Bt gene from bacteria Control Transformed (Bt gene) Genetically engineered plants that are insect resistant Herbicide resistant soybeans Virus resistant transgenic papaya Golden Rice vitamin-enriched bananas The Department of Agriculture has approved genetically modified apples that never turn brown A glowing plant that could provide a sustainable light source Alfalfa transformants with the maize SPS gene driven by a constitutive promoter Non transformants SPS3 SPS6 SPS12 SPS16 SPS1 8 The transformants outperform the non transformed plants significantly SPS 22 In the U.S., genetic screening for certain disorders has become common medical practice For example Pregnant women over 35 years of age are screened routinely to see if they are carriers of chromosomal abnormalities Rates of such defects increase with the age of the mother Widespread screening for phenylketonuria Genetic testing has also been conducted on specific population in which a genetic disease is prevalent Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Genetic testing can be performed in conjunction with in vitro fertilization (IVF) called Preimplantation genetic diagnosis (PGD) IVF involves combining sperm and egg outside of the mother’s body One or two cells are removed at 8 cell stage Tests are done to check for problems Molecular tests can check for particular gene defects Chromosome composition can be checked Decision can be made whether to implant or not. Many ethical questions arise from this process Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Pre-implantation genetic diagnosis Genetic Screening is amongst the newest and most sophisticated techniques used to test for genetic disorders by direct examination of the DNA itself. HUMAN GENE THERAPY Gene therapy is the introduction of cloned genes into living cells in an attempt to cure disease Research efforts in gene therapy is aimed to Alleviate inherited diseases Treat diseases such as cancer and heart disease Combat infectious diseases such as AIDS Human gene therapy is still at an early stage Nevertheless, some of the initial results are promising and future prospects abound *Gene editing Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Pre-implantation genetic diagnosis Gene Therapy Involves the Introduction of Cloned Genes into Human Cells Two transfer methods are used 1. Nonviral approach Liposome technique most common 2. Viral approach Most common are retroviruses, adenoviruses and parvoviruses Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Aerosol Sprays May Be Used to Treat Cystic Fibrosis Cystic fibrosis (CF) is one of the most common recessive inherited disorders Affects about 1 of 3,000 babies of Northern European descent It is caused by a defect in a gene that encodes an ion transport protein This leads to an abnormality in salt and water balance This, in turn, leads to accumulation of mucus in the lungs The result is chronic lung infections which prove fatal Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display CF has been the subject of much gene therapy research To implement CF gene therapy, the normal CF gene must be delivered to lung cells The ex vivo approach used in ADA gene therapy is not possible CF gene therapy has involved the use of an aerosol spray When inhaled by the patient via an aerosol spray, the lung epithelial cells take up liposomes and adenoviruses Still at an early stage of development Eventually may become an effective method of treatment Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Variable-number tandem repeats (VNTRs, or minisatellites) contain repeating clusters of 10-100 nucleotides that are variable between individuals DNA fingerprinting – Forensics DNA fingerprinting – Paternity tests