Transgenic Species and Genetic Manipulation: Transgenic species are organisms which have had genetic material from a different species experimentally transferred into their chromosomes. The transferred gene instructs the transgenic organism to produce the desired trait or product, which may be passed on to future generations. In creating transgenic species, scientists choose the trait they want to express and, using the recombinant DNA technology explained below, isolate the DNA segment and transfer the genes for that trait into the reproducing cells or splice the gene into the DNA of another organism. The Process of Creating Transgenic Species: (This entire process can be seen in the flow diagram “Diagram 1: The Process of Cloning using Recombinant DNA used to produce Transgenic Species” o Scientists choose the trait they want to express and isolate the DNA segment for that trait. o Chemicals called “restriction enzymes” act as the scissors to cut the DNA. Thousands of varieties of restriction enzymes exist, each recognizing only a single nucleotide sequence. Once it finds that sequence in a strand of DNA, it attacks it and splits the base pairs apart, leaving single helix strands (“sticky ends”) at the end of two double helixes. o In this specific case, as well as cutting the DNA segment, a plasmid (small rings of DNA found in bacteria) are also cut using the restriction enzymes to produce sticky ends. o At this stage, scientists are free to add any genetic sequences they wish into the broken chain, usually to give resistance to a particular antibiotic. (This is done so that afterwards, by culturing the bacteria in a medium which contains the antibiotic – only those bacteria that have been successfully transformed will survive.) o After this process, the chain is repaired (as a longer ring of DNA called a “recombinant plasmid”) with another enzyme called “ligase”. o Then, the recombinant plasmid is transferred to a host cell, where the cell’s normal cell division techniques will produce clones of the new DNA sequence. Diagram 1: The Process of Cloning using Recombinant DNA used to produce Transgenic Species (From: “Excel HSC: Biology” [2003 Edition] by Diane Alford and Jennifer Hill) 1. Genetically Modified Foods: The “Flavr Savr” Tomato: The Flavr Savr Tomato was created by Calgene Inc for the purpose of creating a tasty vine ripened tomato that was not destroyed in the process of transportation. The reason why Flavr Savr stays ripe and can stand harsh transportation for at least 10 days after picking (without refrigeration) is through gene manipulation of polygalacturonase (PG), which occurs naturally in all tomatoes. The scientists at Calgene manipulated the genes of the Flavr Savr and suppressed polygalacturonase, which breaks down pectin (found in the cell walls) and prevents the tomato from going mushy. Roundup-Ready Soybeans: In 1995, Monsanto, a large American biotechnology company, created the Roundup-Ready Soybeans. These soybeans have been genetically modified so that they are not affected by Roundup (also owned by Monsanto), which means that farmers can spray the whole field of soybeans with Roundup and not worry that it will also kill the crops. Roundup is made out of a chemical, called glyphosate, which is environmentally friendly as it breaks down easily in water and soil. The theory behind the new Roundup resistant soybeans is the scientists at Monsanto have modified the DNA in the soybean by inserting DNA from soil bacteria, which makes the effects of the glyphosate in the herbicide useless. Hi-Bred Soybeans: In the early 1990s, one of the world's largest seed companies, Pioneer Hi-Bred, developed a more nutritious type of soybean by adding a gene taken from Brazil nuts. The gene coded for a protein rich in methionine, a nutrient that is in short supply in ordinary soybeans. However, the project had to be dropped when it was discovered that the novel soybean would trigger a major attack in people with Brazil nut allergies. Critics often cite this case as an example of one of the major risks of genetically modified food products, particularly as research published a few years previously suggested that this protein was not an allergen. As can be seen from the three case studies above, it is obvious why there is so much controversy over genetically modified foods. It is agreed that some genetically modified foods are better than their natural counterparts, however, such as in the case of the Hi-Bred soybeans, such foods may cause severe allergies or reactions in humans that would not normally be expected in the natural foods. As well, with genetically modified crops, many people are concerned over the possibility of cross-pollination from a genetically modified crop to a non-engineered crop and the effects it could have on the natural products. It is difficult to ensure that this does not occur and spread genes which may, for example, be resistant to pesticides (such as in the case of Roundup-Ready Soybeans) or cause allergic reactions, to their natural equivalent. 2. Some risks that have been suggested in terms of gene technology are: Concerns about the long term affect on the transgenic animal itself, for example, the transgenic pigs which grow much faster and have leaner meat, but are unable to stand due to chronic arthritis. The morality of using animals, with the possibility of giving them painful inflictions. The concern that genetic engineering of transgenic species has the capacity to disrupt the rate of gene transfer between organisms and the way genes are transferred, which therefore disrupts evolutionary relationships between organisms. Concerns that genetically engineered organisms released into the environment may cause new diseases or encourage resistance to current drugs and pesticides. Concerns about the “pollution” of gene pools due to cross-pollination of genetically modified crops to normal non-engineered crops. Concerns about unknown long term health risks associated with eating genetically modified foods or taking genetically modified medicines. 3. Table 1: Reasons For and Against the Development of Transgenic Species: For Against 1. Increases the resistance of 1. Concerns about the long term affect on the transgenic animal plants and animals to itself and the morality of using animals, with the possibility of diseases, pests and extreme giving them painful inflictions. environments. 2. The concern that genetic engineering of transgenic species has 2. Can be used for medicines, the capacity to disrupt the rate of gene transfer between vaccines and to study organisms and the way genes are transferred, which therefore human diseases. disrupts evolutionary relationships between organisms. 3. Improves the productivity of 3. Concerns that genetically engineered organisms released into crops, pastures and animals. the environment may cause new diseases or encourage 4. Increases the efficiency of resistance to current drugs and pesticides. food processing. 4. Concerns about the “pollution” of gene pools due to cross- 5. Improves the quality of food pollination of genetically modified crops to normal non- stuffs. engineered crops. 6. Has the ability to develop 5. Concerns about unknown long term health risks associated new products in many with eating genetically modified foods or taking genetically industries. modified medicines. Artificial Insemination Artificial Insemination is defined as being the introduction of semen to ova through other processes than copulation. These processes usually involve the cell fusion taking place outside the body of the surrogate organism and then being re- introduced back into the uterus and placed for the term of the pregnancy. The process of the most common form of artificial insemination, In Vitro Fertilisation (IVF), is described as follows: (Please also refer to “Diagram 1: Procedure of IVF” for a pictorial explanation of the procedure.) 1. In preparation for the procedure, the female will undergo hormonal therapy, including medication that blocks the secretions of the pituitary gland (thereby, optimising the number of oocytes, or unfertilised ova / eggs, that can be retrieved) and that stimulate ovarian activity, along with blood tests and ultrasound scans of the ovaries to determine the optimal time to retrieve the eggs from the ovary. 2. At the proper time, just before ovulation, a small surgical procedure will allow the female's eggs to be visualised by ultrasound and retrieved from the ovary by placing a fine, hollow needle through the vaginal wall. 3. Then, the embryologist will place the sperm with the eggs when they are ready for fertilisation. Usually, the eggs will develop into cleaving pre- embryos, whose cells divide 2 or 3 times to become pre-implantation embryos (also known as, “pre-embryos”), like the ones shown in Step 5 of Diagram 1. They are maintained in laboratory dishes, in a rich nutrient mixture which acts as a substitute for the environment that would have been provided by the fallopian tubes. 4. Using a special fine catheter, the pre-embryos will then be passed through the vagina and into the uterus at the time the pre-embryos would normally have reached the uterus, approximately two days after retrieval, as shown in Diagram 1 (Step 6). Diagram 1: Procedure of IVF Artificial Pollination Artificial Pollination is defined as being the transmission of pollen onto a stigma by processes that do not occur naturally. This process requires fertilisation using pollen from a selected plant with desirable traits (eg. colour, perfume) and then artificially transferring it to the stigma of another plant. Artificial pollination is, in essence, a reasonably simple procedure to perform: o Firstly, the pollen from the stamen (shown in Diagram 2) of one of the breeding pairs (Plant 1) can be collected using a fine paintbrush. o To ensure that self pollination of the other breeding plant (Plant 2) does not occur, the “male” stamens are removed from the organism. This process is shown below in “Diagram 3: Removing the Stamens from an Angiosperm”. o Then, the pollen from Plant 1 is transferred via hand pollination to the “female” stigma (shown in Diagram 2) of Plant 2 with the cotton swab or artist's brush, or shaken directly over the flowers. Diagram 2: Generalised diagram of the Reproductive Organs in an Angiosperm Cloning Cloning is defined as being the process to make identical copies (known as “clones”) from single cells without fertilisation or the genetical engineering, or the manipulation of the DNA sequencing, of the produced clone. Specifically the term “cloning” may be used to refer to a number of reproductive technologies: o “Animal and Plant cloning” refers to the creation of genetically identical organisms asexually without fertilisation. This can happen in two ways: DNA is extracted from the organism’s tissue and cloned to create an embryo. This embryo is then implanted back into the organism’s uterus for the term of gestation. Cells are taken from an embryo and clones to make multiple copies of the embryo. o “Gene cloning” refers to the creation of multiple copies of DNA segments using recombinant DNA technology, where the DNA segment is isolated and the genes for that trait are transferred into the reproducing cells or “spliced” into the DNA of another organism. This process is used in the hope that scientists can artificially combine the qualities of different organisms; and being able to choose which are the best qualities that particular organism could have. One of the version of plant and animal “cloning” is briefly explained below: o Remove the nucleus of an oocyte (an unfertilised egg) with a micromanipulator (a microscope with fine needles and pipettes to manipulate the oocyte). o Inject the donor nucleus into the oocyte and pulse the egg with a shock of electricity to make the nucleus attach itself to the cytoplasm. This pulse of electricity will begin to “wake” up the cell and make it more likely to start the dividing process. o The resulting cloned ovum can then be re-implanted into a surrogate mother’s uterus to be fertilised to eventually develop into an embryo. 1. Table 1: Potential Risks and Benefits for Cloning Benefits Risks High Risk of Failure: Embryonic stem cells can be grown to produce organs or tissues to repair or replace damaged Cloning animals through somatic cell nuclear ones. Skin for burn victims, brain cells for the transfer is simply inefficient and does not produce brain damaged, spinal cord cells for required results. The success rate ranges from 0.1 – paraplegics, hearts, lungs, livers, and kidneys 3%, which means that for every 1000 tries, could be produced. By combining this approximately only one to 30 clones are made. technology with human cloning technology it Some reasons for this high rate of failure are: may be possible to produce needed tissue for The enucleated egg and the transferred nucleus suffering people that will be free of rejection by may not be compatible their immune systems. Conditions such as An egg with a newly transferred nucleus may Alzheimer's disease, Parkinson's disease, not begin to divide or develop properly diabetes, heart failure, degenerative joint Implantation of the embryo into the surrogate disease, and other genetically based problems mother might fail may be made curable. The pregnancy itself might fail With cloning, infertile couples could have Problems during later development: children. Despite being quite well-known about and publicised in the media, infertility programs Cloned animals that do survive tend to be much are not very successful. (One estimate is that bigger at birth than their natural counterparts. current infertility treatments are less than 10 Scientists usually refer to this as “Large Offspring percent successful.) Human cloning could make Syndrome” (LOS). Clones with LOS have it possible for many more infertile couples to abnormally large organs which can lead to have children than ever before possible. breathing, blood flow and other problems. Since LOS doesn't always occur, scientists cannot reliably predict whether it will happen in any given clone. Also, some clones without LOS have developed kidney or brain malformations and impaired immune systems, which can cause problems later in life. Abnormal gene expression patterns: Lastly, human cloning technology may be able In a naturally-created embryo, the DNA is to reduce the amount of defective genes in our programmed to express a certain set of genes. Later society. (Currently, the average person carries 8 on, as the embryonic cells begin to differentiate, the defective genes inside them.) These defective program changes. For every type of differentiated genes allow people to become sick when they cell - skin, blood, bone or nerve, for example - this would otherwise remain healthy. With human program is different. In cloning, the transferred cloning and its technology it may be possible to nucleus doesn't have the same program as a natural ensure that we no longer suffer because of our embryo. It is up to the scientist to totally reprogram defective genes. the nucleus as complete reprogramming is needed for normal or near-normal development. Incomplete programming will cause the embryo to develop abnormally or fail.