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Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. MCDB 1041 Class 28 Learning Goals: • Compare the creation of a multi-cellular transgenic animal with a transgenic bacterium • Explain the importance of the promoter region in the production of transgenic animals 18-1 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. A piece of DNA that is combined with another piece of DNA not usually associated with it is Recombinant DNA An organism that contains DNA from another organism in its genome is called a Transgenic organism 18-2 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. A transgenic animal has a modified piece of DNA (from another organism, or altered in some way) present in: a. every cell. b. a particular set of cells, determined by the scientist c. a few random cells of the animal 18-3 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. To grow a whole animal that contains the foreign piece of DNA: • Recombinant DNA is injected into a fertilized embryo in a culture dish, before it starts dividing • As a result, all of the cells of the organism will have the genetic alteration present (whether it’s a plant or an animal). A bacterium, since it is a single cell, obviously just contains the altered piece of DNA. 18-4 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. The foreign gene becomes incorporated into one of the chromosomes in the host embryo. (this is a rare and random event) If the transfer occurs, then as embryo grows, all cells within embryo will now have this gene incorporated into a chromosome -Bacterium directly infects a cell (works with plants) -inject recombinant DNA into a fertilized embryo Fig. 14.3b -Use a virus to infect the cell 18-5 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Genetically Modified Foods • • • • higher yield improved quality pest or disease resistance tolerance to heat, cold and drought. Plants have been bred for years to specifically yield these desirable qualities Transgenic technology allows the introduction of genes from OTHER organisms into a plant or animal to yield a specific outcome 18-6 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Insect Resistance Bt corn Normal corn Bacillus thuringiensis (Bt) (a bacterium): Makes a protein that causes paralysis and death to some insects (corn weevil). 18-7 Use this rather than an applied pesticide, which often kills beneficial insects as well! Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Key difference between transgenic bacterium and a transgenic animal (multicellular): You don’t want to spend the time making the transgenic animal only to kill it to isolate the protein for humans! Thus, the animal has to be engineered so that it produces the human protein only in a particular tissue (for example, the cells that produce milk) Then the protein can be harvested. 18-8 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. What controls “expression” of a gene (creation of RNA from the DNA and ultimately production of protein) in a transgenic animal? a. The sequence of the gene itself b. The transcriptional start site c. A promoter region just before the beginning of the gene d. The transcriptional stop sequence at the end of the gene 18-9 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Modifying the expression of a gene To ensure that a recombinant piece of DNA is expressed (translated into protein product) at the right place and time, a specific promoter sequence is added upstream of the coding region This promoter sequence is from a gene which is normally expressed in the tissue of interest. Gene of interest Extract DNA Isolate the gene Add promoter to tell gene where to be expressed promoter Promoter only activated in certain cells So human protein only produced in certain cells 18-10 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Transgenic animals can make human protein • casein is a gene produced in milk • the casein promoter is bound by transcription factors present only in milk producing cells • since this is hooked to the human gene, the human gene will be bound by TFs only in those cells 18-11 Used for treating Pompe disease, heart failure due to accumulation of glycogen A recombinant piece of DNA is made The DNA is injected into an oocyte, then fertilized The rabbit is born and grows up The rabbit produces protein, and that protein can be harvested and given to humans Other examples Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Disease model: Transgenic pigs that have the human Alzheimer's disease-causing allele. Used to study the disease and potential treatments Human drugs: Transgenic goat that produces recombinant human antithrombin III (rhAT) in their milk. This protein prevents blood from clotting. 18-12 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Try this application question: You visit a sheep farm where they engineer sheep to produce human growth hormone. They test the sheep when they are young to determine if they are transgenic. Human growth hormone is 10 KB. Sheep growth hormone is 5 KB. A single set of PCR primers amplify both of these genes from a tissue sample from the sheep. Which of the following lanes correctly represents the DNA from a sheep that is transgenic for human growth hormone? Top of gel (-) A. 10 5 18-13 + B. C. D. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Handout! 18-14