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Transgenics: Glofish

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Hi guys, due to recent discussion here on glofish, I decided to post my research assignment for biology I did earlier in the year. This is not the final copy of the assignment, as it went off into what the assignment was asking. This is just the research and explanations of recombinant DNA for glofish.

Hope you guys find it interesting! The last few paragraphs are meant to be our own opinion on the technology and issues that can arise or are of concern. :) I left everything referenced.

Glofish® , first successfully produced through transgenic modification using zebra danios in 1999 by scientists at the National University of Singapore(Lerner, 2012) were originally designed to help detect environmental pollutants by adding a natural green fluorescence gene found jellyfish to the zebra danios. The aim of the science used to detect pollutants is to one day make the fluorescent gene ‘switch on’ when pollutants and other harmful toxins are present within the environment. (ww.glofish.com, 2010)

Zebra Danios’ (Danio rerio) were used to make Glofish® due to their valuable features that make them a model organism. Such features are:

- Their easy ability to breed

- Ease to maintain in an aquarium environment

- Produces a large number of offspring

- Eggs are transparent, allowing for easy insertion of genes, as well as easy observation of developing organs after fertilisation

, n.d.)

As Glofish® have become one of the first transgenic animals allowed for sale to the public, they underwent testing by the Food and Drug Administration (FDA) in the US to regulate genetically modified organisms to ensure the safety of the public, the environment, as well as the fish used prior to sale. Studies carried out by the FDA proved that even if Glofish® were to be released into the wild, it would be very difficult for them to survive due to their captive upbringing, their personal water chemistry requirements, as well as their ability to fluoresce, making them unable to blend in well to the surrounding environment. (Lerner, 2012)

The science that has allowed Glofish® to fluoresce is a form of genetic modification called Transgenics (also known as recombinant DNA) which first emerged in the 1960’s, with the basic principles of recombination been discovered previously around 1928. Transgenics is essentially the transfer of a desired gene (such as the fluorescent protein of jellyfish) from one organism to another organism such as the zebra danios. Often, selective breeding is needed in order to notice the effects, or have the transgenic gene present and active within the animal. (Pray, 2008)

Recombinant DNA technology: Creating transgenic animals

Recombinant DNA (rDNA) is a form of artificial DNA created as a result of combining desired DNA from one organism into another, creating a combination that would not otherwise occur naturally. The cut and paste method, commonly used to produce Glofish® involves using enzymes to aid in cutting and inserting a desired gene from one genome into another by attaching using DNA ligase as a bind resulting in rDNA. (users.rcn.com, 2011)

550204_488986361122330_226539364_n.jpgFigure 3 Foreign gene attaching to DNA of another organism (www.microrao.com, 2006)

The process in which the cut and paste method is used to create transgenic animals first began when American scientists were able to purify and characterise an enzyme found in E.Coli that acted as a biological glue to attach DNA together. This Biological glue known as DNA ligase could further be used to attach foreign DNA together. (Pray, 2008) This sort of biological glue is used in all organisms during replication of DNA, as DNA cannot be attached without a version of DNA ligase. In order to DNA to be attached, the nucleotide order in which DNA is formed must be cut correctly for the foreign DNA to be able to attach whilst still remaining in the correct chemical base sequence. (Klose, Lampard, 2010) This can be seen in Figure 3, which shows that the blue gene showing the sequence GAATTC and CTTAAG must attach to the foreign DNA where the sequence will be in the correct order.

541096_488986341122332_319509423_n.jpgFigure 2 Combination of forgien DNA with a plasmid (Pray, 2008)

In order for genes to be cut out of a DNA sequence, restriction enzymes are needed to precisely cut the DNA to expose the desired gene. Restriction enzymes are found within bacteria that act as biological scissors. For the process of producing transgenic animals, restriction enzymes are sequence specific, and can be used to cut desired sequences that make up a specific gene (the desired gene) within the DNA. In order for a desired gene to be cut and removed( such as the fluorescent protein gene in jellyfish), specific restriction enzymes bind to the desired DNA sequence, and digest the sugar-phosphate binds which are essentially the backbones of DNA strands, leaving overhangs known as ‘sticky ends’.(www.dnalc.org, n.d.)

(Refer to figure 2) In order to effectively transfer genes into cells, a circular DNA molecule known as a plasmid is used as a vector. Plasmids are found in some bacteria, and easily multiply, making them effective ‘vehicles’ to transfer genes from one cell to another. (Pray, 2008) Plasmids must also be cut using the same restriction enzyme used to isolate a specific gene in order to expose an area in which the gene is able to combine with the plasmid. Both the plasmid and isolated gene are left with overhangs (sticky ends). These overhangs are essential in combining DNA as they are able to base pair with any DNA molecule that contains the complementary overhangs. The Plasmid and foreign DNA are then connected, and DNA polymerase (Enzyme that catalysed the polymerisation of nucleotides into a DNA strand) is then added to insert any missing nucleotides that may have occurred. DNA ligase is then added, which seals and re-attaches the sugar-phosphate binds resulting in a molecule of rDNA. (www.dnalc.org, n.d.)

After the rDNA has been created, the recombinant DNA is cloned and introduced into an animal cell via transformation. There are several ways of transformation, however in order to produce transgenic animals, the most commonly used method of transformation is called microinjection. (Klose, Lampard, 2010)

Microinjection occurs by injecting a fertilised egg with the new rDNA, which is then replicated and becomes present in the genetic make-up of a forming embryo, producing a transgenic animal. (www.glofish.com, 2010)

Genetic modification has allowed for the production of healthier food products, medical advancement, human health, as well as animal health, and has become a very beneficial science within today’s society. Genetic modification, particularly the production of transgenic animals has allowed for the production of things such as insulin for humans suffering from diabetes, milk containing particular human proteins, antibodies, as well as models for the treatment of diseases in animals and in humans. (www.actionbioscience.org, 2010)

The technology used to produce transgenic animals such as the cut and paste method allows for the production of animals with desired genes of other animals. This science not only can benefit the overall health of the environment such as what Glofish® are designed to do, but also aid in preserving animals within the environment that may be endangered or facing extinction. Similarly to Glofish®, glow-in-the-dark rabbits have also been produced using the same technology in order to help track cells throughout the body to monitor the success or failure of an organ transplant. Furthermore, this technology used on the rabbits can then be used to track cancer cells within humans and animals, potentially saving thousands of lives.

Despite the fact that this science can positively benefit the world of science, the environment, and the welfare of animals and humans, there is also room for concern due to the misuse of the technology. One major concern of the misuse of the technology is the effect transgenic animals pose on the environment

As transgenic animals do not occur naturally, animals that have been modified transgenically raise the concern of being released into the wild, causing devastating environmental impacts. For example, animals made to show different colours that may be more vibrant or aesthetically appealing have the potential to be released into the wild, breeding, and becoming more recognisable to prey or predators which can result in the abundance and/or decrease of animal populations within the environment.

Another concern brought up through the incorrect use of the technology is producing animals for the sake of commercial use, which can pose adverse effects on the animal’s gene pool. Considering Glofish® now being a commercially available animal, companies as well as scientific institutes have the option to create genetically modified animals for the sake of commercial use. Although transgenic animals must be approved before production, as well as be approved for commercial sale and use, the impacts transgenically modified animals pose in the future is still in the preliminary stages of research, meaning that transgenically modified animals allowed for commercial use pose the risk of accelerating genetic erosion of animals that have not been genetically modified. (buzzle.com, 2012)

Even though scientists have the technology to transgenically modify animals, whether or not they should do so is very debatable, and presents areas for concern. Ethically speaking, using animals for genetic engineering can put animals at risk of pain and suffering, and the welfare of any animal used for genetic engineering needs to be taken into account. As a personal viewpoint to the technology, transgenic animals should not be created for the purpose of commercial sale or use, and should not be used to create animals with diseases in order to trail new drugs intended for human consumption. Not only is this an ethical issue, but transgenically modified animals made for the purpose of showing human diseases cannot provide a completely accurate report on whether the treatment used on the modified animal will have the same effects once used on humans. As animals do not represent the whole of the human body to biological differences, there is no way in which drugs can be guaranteed to have the same effects on different species. Other methods in which drugs can be successfully tested should be used instead, such as testing cells, cell cultures, and even organ slices that are species specific.

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