This page was produced as an assignment for Genetics 677, an undergraduate course at UW - Madison.
Researching the Effects of PAH
The PKU phenotype refers to the characteristics of a person with PKU. Phenotype can refer to the outward symptoms (such as mental retardation, pale coloring, epilepsy, etc.), but it can also refer to the microscopic interactions and goings-on. Therefore, a person may have the PKU phenotype in the sense that they have non-functional PAH protein, but because of prevention efforts or lifestyle choices, they may not show most, if any, of the outward and obvious symptoms of the disease. No matter what types of symptoms (outward, microscopic, or both) are being researched, the right type of organism must be chosen to use for experiments. Using large organisms can allow for easy visualization and observations of outward effects. Research on microscopic effects can use samples of the tissue from the chosen organism, which are then analyzed using techniques such as microarrays.
RNAi and Model Organisms
Because experimentation is not always possible on humans, it is important to find other organisms that can take the place of humans. Common choices include mice, rats, zebrafish, and nematodes. There are many factors to take into consideration when choosing a model organism, including cost, generation time, and most essentially, phenotypic similarity. For experiments involving biochemical interactions, it is sometimes possible to study human diseases in organisms as simple as bacteria or yeast, but if the outward and more complex symptoms are of interest, animals that display the same outward symptoms are necessary.
One common way of testing similarity in symptoms is to perform RNAi experiments. In a nutshell, RNAi prevents the protein of a certain gene from being made: it more or less destroys or masks the function of the protein, giving an otherwise healthy animal the diseased state without actually altering the genome. Using various RNAi databases, one can use information gained from past experiments in order to decide which organism to use for current research.
Using the RNAi databases for nematodes, flies, zebrafish, and mice, it became apparent that mice are the ideal model organisms for studying PKU. Knocking out PAH expression in embryonic lethality in nematodes, light sensitivity and disrupted musculoskeletal movement in zebrafish, and varying defects in coloring (and sometimes embryonic lethality) in flies [1,2,4]. Knockouts in mice revealed higher-than-usual blood and urine Phe levels, microcephaly, cognitive defects, seizures, and lighter-than-normal pigmentation [3]. Clearly, the phenotype expressed in mice is nearly identical and much closer to the human phenotype. Though they are more expensive to raise and care for, it is important to perform experiments on organisms that will give the best live representation as to how a human would react to the same treatment. Therefore, mice are the clear choice in model organism for studying PKU.
One common way of testing similarity in symptoms is to perform RNAi experiments. In a nutshell, RNAi prevents the protein of a certain gene from being made: it more or less destroys or masks the function of the protein, giving an otherwise healthy animal the diseased state without actually altering the genome. Using various RNAi databases, one can use information gained from past experiments in order to decide which organism to use for current research.
Using the RNAi databases for nematodes, flies, zebrafish, and mice, it became apparent that mice are the ideal model organisms for studying PKU. Knocking out PAH expression in embryonic lethality in nematodes, light sensitivity and disrupted musculoskeletal movement in zebrafish, and varying defects in coloring (and sometimes embryonic lethality) in flies [1,2,4]. Knockouts in mice revealed higher-than-usual blood and urine Phe levels, microcephaly, cognitive defects, seizures, and lighter-than-normal pigmentation [3]. Clearly, the phenotype expressed in mice is nearly identical and much closer to the human phenotype. Though they are more expensive to raise and care for, it is important to perform experiments on organisms that will give the best live representation as to how a human would react to the same treatment. Therefore, mice are the clear choice in model organism for studying PKU.
Microarrays
Microarrays are used to quantify and analyze levels of gene expression in organisms that are exposed to certain stimuli or environments. In terms of PKU, someone who is interested in changes in gene expression due to the disease could take tissue/RNA samples from a healthy mouse (or human) and use microarrays to compare healthy expression to the expression found in mice (or humans) with PKU.
To the left is a sample of what a microarray may look like. Green dots represent a gene that is being expressed in one sample (a healthy mouse, for example). Red dots represent a gene that is being expressed in the other (a PKU mouse), and yellow dots are genes that are being expressed in both samples. Microarrays provide an efficient solution to identify possible genes of interest for studying. They can also provide a whole-body view of how a disease affects an individual [5].
There are large databases of microarrays, such as GEO, that have been done that represent various animals, tissues, and disease states. Interestingly enough, microarrays that have been performed on PKU-inflicted animals are extreme few and far between, and surprisingly non-comprehensive. Metabolic disorders can have huge impacts on multiple systems of the body, both obvious and subtle, and microarrays would be an invaluable resource in determining just how these diseases (such as PKU) can affect the body as a whole.
To the left is a sample of what a microarray may look like. Green dots represent a gene that is being expressed in one sample (a healthy mouse, for example). Red dots represent a gene that is being expressed in the other (a PKU mouse), and yellow dots are genes that are being expressed in both samples. Microarrays provide an efficient solution to identify possible genes of interest for studying. They can also provide a whole-body view of how a disease affects an individual [5].
There are large databases of microarrays, such as GEO, that have been done that represent various animals, tissues, and disease states. Interestingly enough, microarrays that have been performed on PKU-inflicted animals are extreme few and far between, and surprisingly non-comprehensive. Metabolic disorders can have huge impacts on multiple systems of the body, both obvious and subtle, and microarrays would be an invaluable resource in determining just how these diseases (such as PKU) can affect the body as a whole.
References
1. WormBase web site, http://www.wormbase.org. From: http://www.wormbase.org/species/c_elegans/gene/WBGene00000240#0a-9e-3
2. Zebrafish Information Network (ZFIN), University of Oregon, Eugene, OR 97403-5274; World Wide Web URL: http://zfin.org/ZDB-GENE-031006-2 [04/21/2013].
3. Mouse Genome Database (MGD), Mouse Genome Informatics, The Jackson Laboratory, Bar Harbor, Maine. World Wide Web (URL: http://www.informatics.jax.org). (April, 2013).
4. S.J. Marygold, P.C. Leyland, R.L. Seal, J.L. Goodman, J.R. Thurmond, V.B. Strelets, R.J. Wilson and the FlyBase Consortium (2013).
FlyBase: improvements to the bibliography. Henna.
5. "Just the Facts: A Basic Introduction to the Science Underlying NCBI Resources." NCBI. U.S. National Library of Medicine, 27 July 2007.
2. Zebrafish Information Network (ZFIN), University of Oregon, Eugene, OR 97403-5274; World Wide Web URL: http://zfin.org/ZDB-GENE-031006-2 [04/21/2013].
3. Mouse Genome Database (MGD), Mouse Genome Informatics, The Jackson Laboratory, Bar Harbor, Maine. World Wide Web (URL: http://www.informatics.jax.org). (April, 2013).
4. S.J. Marygold, P.C. Leyland, R.L. Seal, J.L. Goodman, J.R. Thurmond, V.B. Strelets, R.J. Wilson and the FlyBase Consortium (2013).
FlyBase: improvements to the bibliography. Henna.
5. "Just the Facts: A Basic Introduction to the Science Underlying NCBI Resources." NCBI. U.S. National Library of Medicine, 27 July 2007.