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Name: ______________________ Date: ________________ Glowing Fish - First Genetically Modified Organism Available as Pets Genetic Engineering in Animals The first genetically modified organism to be made available as a pet was the GloFish. The GloFish was essentially a zebrafish that had been modified with bright red, green and orange fluorescent colors. Like other genetically modified organisms, the GloFish was merely an experiment, but it soon grew to prominence on the ornamental fish market (a market that sells fish based on appearance). GloFish Development Zebrafish are native to rivers in India and Bangladesh. They measure approximately three centimeters long and feature gold and dark blue stripes. However, natural zebrafish have troubles breeding in the United States due to the fact that they are tropical fish. This means they cannot survive in the wild. These facts played heavily into the questions regarding the pros and cons of genetic engineering of ornamental fish. While working at the National University of Singapore in the late 1990s, Dr. Zhiyuan Gong and his associates extracted a protein from a jellyfish and inserted the gene into the zebrafish genome. What does this mean, you ask? We can start by stating that each organism has an instruction manual, or chemical blueprint, in each one of his or her cells. That instruction manual is called DNA. Each “page” of the instruction manual contains steps to build a different part of the organism. Each “page” or segment of DNA, is called a “gene”. In simpler terms, Dr. Gong removed the gene, or chemical instructions, that enable jellyfish to glow, and stuck it into the DNA of a zebrafish, creating a transgenic organism. The purpose of this experiment was to produce a fish that could be used to detect pollutants and environmental toxins. The development of a fluorescent fish needed to be completed first in order to continue the experiment. The goal was to create fish that would “glow” in the presence of toxins. Gong and his team worked very hard to with zebrafish, and created multiple versions of these glowing fish. Some contained jellyfish DNA while others contained genes from sea coral giving them a red and yellow color. The fluorescent genes are extracted from jellyfish or sea coral and then injected into zebra fish eggs. The genes were then inserted into the DNA found within the zebra fish eggs. With these newly inserted genes, the body of the zebra fish is now capable of giving off a fluorescent glow. Scientists also added an “on-andoff” switch made of DNA, that cause the Glow-gene to be turned on or off in certain conditions. The on/off switch is called a promoter. So far, the researchers have succeeded in isolating two types of gene promoters in the zebra fish an estrogen-inducible promoter and a stress-responsive promoter. These promoters have been used to drive the fluorescent color genes in transgenic zebrafish. Such fluorescent-colored transgenic fish will be able to respond to the presence of chemicals like estrogen through the estrogenic promoter and heavy metals and toxins through the stress-responsive promoter. The fish will immediately display the color depending on the type of environment the color has been specified for. For example, if a transgenic zebra fish is swimming in waters that contain estrogen (a female hormone that promotes female sex characteristics), the estrogen-inducible promotor will be activated and the fish will begin to glow. Techniques for generating Transgenic Organisms The three principal methods used for the creation of transgenic animals are DNA microinjection, embryonic stem cell-mediated gene transfer and retrovirusmediated gene transfer. a) DNA microinjection. This method involves injecting a chosen gene, or genes, from one organism into the egg of another. The gene can be transferred from an organism of the same species, or an entirely different species. It is one of the first methods that proved to be effective in mammals and in many other types of species (Gordon and Ruddle, 1981). When the new gene is inserted into the hosts DNA it will either cause the hosts cells to produce more or less of the new protein or a protein that already exists in the host cell. The insertion of DNA is, however, a random process, and there is a high probability that the introduced gene will not insert itself into a site on the host DNA that will allow it to be expressed. Once the egg is made it will be transferred to female recipient that will act as a surrogate mother. Sometimes the act of mating is vital for the female body to change and nurture the growth of a developing embryo. To simulate this act often time’s scientist will use male zebra fish that have been vasectomized. b) Embryonic stem cell-mediated gene transfer. (Knockout Method) Stem cells are undifferentiated cells that have the potential to develop, or differentiate, into any type of cell (neuron, heart cell, eye cell, skin cell, etc.) In the stem cell method, the transgene (gene from the foreign organism) is inserted into a colony of stem cells in a Petri dish. These cells are then incorporated into an embryo when the embryo is a small ball of cells. The result is a chimeric animal, meaning some of the cells in the organism have the transgene in them, and some of them are “normal” without the transgene. The challenge with this is that not every cell has the gene of interest. In order to get every cell of an organism to have this transgene you would have to breed them for a minimum of 10-20 generations before you made a fully transgenic organism. c) Retrovirus-mediated gene transfer. Viruses work by taking over the cell, and forcing the cell to replicate the virus. Essentially, viruses hijack the cell and use them to make more viruses (and then invade other cells). Retroviruses are a kind of virus that can be used to transfer a transgene into the (hopefully) newly transgenic organism. Scientists insert the transgene of interest into the virus’s genetic material, and the virus does the rest: enters some of the cells of an embryo, inserts itself into the DNA of the organism, and the cell begins to express the gene. Offspring derived from this method are chimeric, i.e., not all cells carry the retrovirus. The challenge with this is that not every cell has the gene of interest. In order to get every cell of an organism to have this transgene you would have to breed them for a minimum of 10-20 generations before you made a fully transgenic organism. For any of the above techniques the success rate in terms of live births of animals containing the transgene is extremely low. Providing that the genetic manipulation does not lead to abortion, the result is a first generation of animals that need to be tested for the expression of the transgene. Depending on the technique used, the first generation may result in chimeras. Questions: Use the article to answer the questions. 1. What is a zebrafish? 2. What is a glofish? 3. What is a gene? 4. What is a promoter? 5. What is a transgenic organism? 6. Explain how scientists created glofish from zebrafish? 7. Explain how scientists used glofish to study water pollution before they became commercially available for use as pets? 8. What is estrogen? Why wouldn’t it be good to have it in water supplies? 9. In your opinion, are there ethical issues surrounding genetically modified glofish? Explain. 11. How does this article relate to the movie “Avatar”? 12. How will this article help you answer the question, “How do you create an Avatar?” 13. Why did scientists use the DNA microinjection method, rather than the other methods? Explain.