|MadSci Network: Genetics|
The jellyfish gene used to fluorescently label transgenic animals is the GFP (Green fluorescent protein) gene from the jellyfish Aequorea victoria. The 'fluorescent bunny' web page (which looks like a hoax) has this brief, accurate information on GFP: 'After green fluorescent protein (GFP) was first isolated from Aequorea victoria and used as a new reporter system (see: Chalfie, M., Tu, Y., Euskirchen, G., Ward, W., Prasher, D. (1994). Green Fluorescent Protein as a Marker for Gene Expression. Science 263, 802-805) it was modified in the laboratory to increase fluorescence. See: Heim, R., Cubitt, A. B. and Tsien, R.Y. (1995) Improved green fluorescence. Nature 373:663-664; and Heim, R., Tsien, R. Y. (1996). Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer. Current Biology 6, 178-182. Further work altered the green fluorescent protein gene to conform to the favored codons of highly expressed human proteins and thus allowed improved expression in mammalian cells. See: Haas, J, Park, EC and Seed, B. (1996). Codon usage limitation in the expression of HIV-1 envelope glycoprotein. Current Biology 6: 315-24. More recently, new mutations with greater fluorescence have been developed. See: Yang, Te-Tuan et al. (1998). Improved fluorescence and dual color detection with enhanced blue and green variants of the green fluorescent protein. The Journal of biological chemistry, V. 273, N. 14, p. 8212. For a comprehensive overview of green fluorescent protein as a genetic marker, see: Chalfie, Martin. Kain, Steven. Green fluorescent protein : properties, applications, and protocols (New York : Wiley-Liss, 1998). Since its first introduction in molecular biology, GFP has been expressed in many organisms, including bacteria, yeast, slime mold, many plants, fruit flies, zebrafish, many mammalian cells, and even viruses. Moreover, many organelles, including the nucleus, mitochondria, plasma membrane, and cytoskeleton, have been marked with GFP.' This is quoted from: http://www.ekac.org/ gfpbunny.html By the way, I think that this site is a hoax because it was put up by a sort of performance artist and the picture of the green bunny is obviously fake. It wouldn't surprise me if there were some GFP rabbits (there are certainly transgenic rabbits). Sorry if this wasn't the story that you were referring to. To explain GFP a little, proteins are polymers of amino acids. Twenty different amino acids are used in the synthesis of proteins. These amino acids differ in the chemical nature of their side chains, which can range from a simple hydrogen atom to a complex double-ring structure. Some of the amino acids are somewhat fluorescent, and depending on the exact sequence of the protein and the way the protein folds, some proteins are quite fluorescent. The GFP protein is a really striking example of a protein that is naturally fluorescent without having chemical dyes attached to it. The gene for GFP has been modified to make it more fluorescent, and to change the color of the fluorescence. Modified GFP genes are available commercially. See, for example: http://www.clontech.com/gfp/ There are pictures of GFP-expressing tissue culture cells, zebrafish, mosquitos and mice available in the Clontech GFP brochure, which you can view as a PDF file. Some variant of GFP was used to make ANDi. The paper describing the creation of ANDi is: Chan AW, Luetjens CM, Dominko T, Ramalho-Santos J, Simerly CR, Hewitson L, Schatten G Foreign DNA transmission by ICSI: injection of spermatozoa bound with exogenous DNA results in embryonic GFP expression and live rhesus monkey births. Molecular Human Reproduction 2000 6:26-33. Oregon Regional Primate Research Center, Departments of Obstetrics, Gynecology and Cell Developmental Biology, Oregon Health Sciences University, 505 NW 185th Avenue Beaverton, OR 97006, USA. These researchers used a technique called intracytoplasmic sperm injection. In this technique, DNA consisting of a retroviral vector containing the GFP gene was used to coat rhesus monkey sperm. The sperm was microinjected into rhesus monkey eggs to create embryos. The technique of intracytoplasmic sperm injection (ICSI) is used in human fertility clinics as a method of creating human embryos for implantation in women. In fertility clinics, of course, the sperm are not coated with foreign DNA. When the DNA-coated rhesus monkey sperm was injected into the egg, it fertilized the egg to create a monkey embryo, but also allowed the retrovirus to infect the embryo cells and cause copies of its genetic material, including the GFP gene, to be inserted randomly into the monkey chromosomes. Earlier work by these researchers showed GFP expression (meaning synthesis of the GFP protein) in embryo cells. Some live births resulted from embryo implantation, but none of the monkeys carried the GFP gene in their chromosomes in the earlier work. There are a number of techniques besides ICSI to generate transgenic animals. In mice, two general sorts of techniques are used. One is the injection of foreign DNA into one-cell mouse embryos. In this technique, mouse eggs are fertilized in vitro. A glass microneedle and micromanipulator apparatus is used to inject a small amount of a DNA solution into the nucleus of a one-cell embryo. The transgene DNA might be in a retroviral vector, which would allow a copy of the gene to transfer itself to the mouse chromosomes as the monkey transgene did. It can also be in a 'conventional' cloning vector like a bacterial plasmid; somehow, this sort of DNA can integrate into the mouse chromosomes using the same enzymes in the cell that repair damage to DNA. The problem with making transgenic mice (or monkeys) this way is that the DNA might integrate anywhere in the chromosomes, including in the middle of an important gene. Researchers are also not able to control the number of copies that integrate, and transgenic animals produced this way generally have multiple copies of the transgene. For many experiments, it wouyld be better to have a single copy at a known chromosomal location. The other technique for making transgenic mice is called 'targeting'. This technique has not yet been extended to other mammals, but it could be. In this method, a special cell line derived from mice is used. The cell line (cells that can be grown in tissue culture) is called an embryonic stem cell (ES cell) line. These cells can be grown in a special medium in dishes in an incubator, but have the property that they can be incorporated into mouse embryos and differentiate into all kinds of mouse tissues, including the germ line (the cells that give rise to sperm and eggs). To make a chimeric embryo, a mouse embryo at the blastocyst stage is mixed with ES cells, and the aggregate is implanted into a foster mother. The ES cells are from a mouse strain that has light fur. The blastocyst is usually taken from a strain that has black fur. This way, when chimeric mice are born, they can be recognized by patches of black and light fur. These are bred to other mice, and if the ES cells are part of the germ line, the genes of the ES cells will be passed on to the progeny. To make transgenics using this technique, the ES cells are transformed (that is, they take up DNA) using a very special DNA construct. DNA from the gene to be modified in the ES cells is incorporated into a DNA construct using recombinant DNA techniques. If you imagine the gene as having the sequence A-B-C, a construct is made that has the sequence A-C. This DNA is put into the ES cells, where it integrates into the mouse chromosomes by a process called recombination. This process uses enzymes that ordinarily repair DNA based on matching similar sequences using base pairing. The recombination event replaces one of the two chromosomal copies of the gene with the sequence A-B-C with the construct that has the sequence A-C. Since a piece of the gene is missing, the gene usually doesn't work anymore. The mice are OK because they have a second copy. When the progeny are inbred, some embryos are made that have two copies of the 'knock-out' (as it is called). These might die as embryos if the gene is important for development, or they might be born as mice that have a mutant phenotype. This technique is very useful, because the normal copy of the gene is replaced with a copy that has the sequence desired by the experimenter. Before the transformed ES cells are used to make mice, the experimenter verifies that the modified gene has replaced one of the chromosomal copies. Usually a gene making the cells resistant to a toxic drug is added to the construct. That way, it is possible to kill all the ES cells that didn't corporate the gene by adding the drug to the tissue culture medium. Here is a site with a cartoon showing the creation of mouse knockouts: http:// www.ri.bbsrc.ac.uk/molbiol/mcwhir/embryoni.htm I hope that this explanation is helpful to you. It is very hard to write about this subject without using genetics terms. We have an online glossary of such terms, with links to pictures, in our database. You can see our Glossary at: http:// www.informatics.jax.org//userdocs/glossary.shtml Yours, Paul Szauter Mouse Genome Informatics
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