This web page was produced as an assignment for Genetics 564, an undergraduate course at UW-Madison
Mutant Phenotypes in Model Organisms
Model organisms have been used for decades in research as a way to learn about diseases and processes within the body without having the ethical concerns associated with human experimentation. There are several organisms whose internal systems are analogous to humans, but they reproduce faster, are easy to obtain, and easier to manipulate. Mutant strains can be created by gene knockout or mutagenic agents, and the resulting organisms can be used to see what phenotypes are present when certain genes are disrupted [1]. Oftentimes, scientists find that the best way to study a gene is to see what happens when it is defective. They are able to carry out these types of experiments with model organisms.
Analysis
The zebrafish database ZFIN was used to find mutants in the slc6a4a gene. The MO1-slc6a4a knockout strain, created by slice-blocking MO, is available for study from this group. While there is no data available about the gene expression for these knockout zebrafish, there is a notable phenotypic effect on the skeletal myofibril such that it appears disorganized and doesn't function as well as wild-type [2]. It has been studied in the context of Duchenne muscular dystrophy [3].
IMSR, the International Mouse Strain Resource, listed fourteen mutated strains of mice involving the Slc6a4 gene. Two of these strains, Slc6a4tm1Kpl and SERT-KO, are both available for order from the Jackson Laboratory and Klaus-Peter Lesch respectively. Increases in anxious behaviors are seen in both strains, with the first implicated in the study of OCD [4] and the second in autism [5]. Behaviors include decreased aggression, elevated responses to minor stressors, and decreased exploration of new environments or objects. There is no effect on fertility or viability, though the anxious behaviors may lead to poor mothering from parental mice [6,7].
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The main database for finding Drosophila mutants is FlyBase. One RNAi regulatory mutant related to the fly SerT gene is SerTGD3824. There are no visible phenotypes and no notation in the Bristle Screen Database, but the mutation is viable and reproduction is not affected. Very little research has been done on this mutant.
WormBase is commonly used to find variants for Caenorhabditis elegans. C. elegans is not a homolog for the SLC6A4 protein in humans, as indicated by a BLAST and reverse BLAST search. MOD-5 is, however, an ortholog and therefore does have some underlying relationship to the SLC6A4 protein. The n822 variant of MOD-5, created through substitution and available from CGC Genetics, has three distinct phenotypes [8]. First, They are antibody staining variants, as evidenced by Kullyev et al [9]. They are also desensitized to NaCl, such that the signals from the ions are not as effective [10]. n822 variants are shown to be drug resistant to fluoxetine (Prozac) to some degree compared to the wild-type organisms as well [9].
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Discussion
One notable point of discussion is the use of RNAi mutants versus knockout mutants. While RNAi is intended to knock out genes through different mutations and inversions within the appropriate segment of the genome, it is often incomplete. As such, the phenotype that is observed in these organisms is often muted [11]. In the case of this study, this may explain why the fly model showed no phenotypic alteration while the other models did. Because the phenotypes are often behavioral, the subtle difference between the complete knockdown and RNAi mutants may be enough to mask the phenotypic expression in the latter.
Most strongly implicated in its relationship to OCD are the mouse strains. The behavioral expressions in both variants are on par with the phenotypes in an anxiety-related disorder. The lack of change to fertility or lethality status in the mutant strains have also made these organisms very easy to work with and manipulate. Researchers have made this connection, and at this point the most common model organism to study mood and anxiety disorders is the mouse or rat.
Though it is less obvious, C. elegans mutant n822 is related to human variants in its sensitivity to ions. In Homo sapiens, the SLC6A4 protein is dependent on ion concentrations including sodium, potassium and chloride. Likewise, the MOD-5 protein is sensitive to concentrations of sodium and chloride in order for it to function correctly. When the substitution mutation is present in the n822 strain, there is desensitization to these signals. Along with this, there is slight drug resistance to fluoxetine, and SRI, in the n822 variants. As SRIs are the most common drug-based therapies for OCD, this resistance is something that should be studied more in the future to see if more effective pharmaceuticals can be developed.
Zebrafish have unique phenotypic responses to serotonin transporter gene alteration, showing abnormalities in their skeletal myofibril organization and function. The study by Waugh et al., which made this phenotypic characterization, suggests a direct correlation between serotonin and muscular function. Though this has not been classically associated with serotonin, there are changes seen in muscle metabolism when serotonin is present and the Waugh et al., study now suggests an effect in fibril membrane stability as controlled by calcium levels. Because the SLC6A4 protein in Homo sapiens is dependent on several ions, it is possible that slc6a4a mutants respond poorly to signals from calcium ions just as the C. elegans mutant is desensitized to sodium and chloride.
Most strongly implicated in its relationship to OCD are the mouse strains. The behavioral expressions in both variants are on par with the phenotypes in an anxiety-related disorder. The lack of change to fertility or lethality status in the mutant strains have also made these organisms very easy to work with and manipulate. Researchers have made this connection, and at this point the most common model organism to study mood and anxiety disorders is the mouse or rat.
Though it is less obvious, C. elegans mutant n822 is related to human variants in its sensitivity to ions. In Homo sapiens, the SLC6A4 protein is dependent on ion concentrations including sodium, potassium and chloride. Likewise, the MOD-5 protein is sensitive to concentrations of sodium and chloride in order for it to function correctly. When the substitution mutation is present in the n822 strain, there is desensitization to these signals. Along with this, there is slight drug resistance to fluoxetine, and SRI, in the n822 variants. As SRIs are the most common drug-based therapies for OCD, this resistance is something that should be studied more in the future to see if more effective pharmaceuticals can be developed.
Zebrafish have unique phenotypic responses to serotonin transporter gene alteration, showing abnormalities in their skeletal myofibril organization and function. The study by Waugh et al., which made this phenotypic characterization, suggests a direct correlation between serotonin and muscular function. Though this has not been classically associated with serotonin, there are changes seen in muscle metabolism when serotonin is present and the Waugh et al., study now suggests an effect in fibril membrane stability as controlled by calcium levels. Because the SLC6A4 protein in Homo sapiens is dependent on several ions, it is possible that slc6a4a mutants respond poorly to signals from calcium ions just as the C. elegans mutant is desensitized to sodium and chloride.
References
Zebrafish Database: http://zfin.org/
IMSR: http://www.findmice.org/index
FlyBase: http://flybase.org/
Bristle Screen Database: https://bristlescreen.imba.oeaw.ac.at/
WormBase: http://www.wormbase.org/#01-23-6
CGC Genetics: http://www.cgcgenetics.com/cgc/en/main-en.html
[1] ModelOrganisms - Home. (2012, January 1). Retrieved March 26, 2015, from http://www.modelorganisms.org/
[2] ZFIN Morpholino: MO1-slc6a4a. (n.d.). Retrieved March 26, 2015, from http://zfin.org/action/marker/view/ZDB-MRPHLNO-141031-1
[3] Waugh, T., Horstick, E., Hur, J., Jackson, S., Davidson, A., Li, X., & Dowling, J. (2014). Fluoxetine prevents dystrophic changes in a zebrafish model of Duchenne muscular dystrophy. Hum. Mol. Gen., 23(17), 4651-4662. Retrieved March 26, 2015, from http://zfin.org/cgi-bin/webdriver?MIval=aa-pubview2.apg&OID=ZDB-PUB-140513-91
[4] Holmes, A., Lit, Q., Murphy, D.L., Gold, E., Crawley, J.N. (2003). Abnormal anxiety-related behavior in serotonin transporter null mutant mice: the influence of genetic background. Genes Brain Behav., 2(6):365-80. Retrieved March 26, 2015, from http://www.ncbi.nlm.nih.gov/pubmed/14653308?dopt=Abstract
[5] Bengel, D., Murphy, D., Andrews, A., Wichems, C., Feltner, D., Heils, A., ... Lesch, K. (1998). Altered brain serotonin homeostasis and locomotor insensitivity to 3, 4-methylenedioxymethamphetamine ("Ecstasy") in serotonin transporter-deficient mice. Mol Pharmacol., 53(4), 648-655. Retrieved March 26, 2015, from http://molpharm.aspetjournals.org/cgi/pmidlookup?view=long&pmid=9547354
[6] JAX Mice Database - 008355 B6.129(Cg)-Slc6a4/J. (2009, November 24). Retrieved March 26, 2015, from http://jaxmice.jax.org/strain/008355.html
[7] EMMA strain search: B6.129-Slc6a4tm1Kpl/Cnrm. (n.d.). Retrieved March 26, 2015, from https://www.infrafrontier.eu/search?keyword=EM:05002
[8] N822 (variation). (n.d.). Retrieved March 27, 2015, from http://www.wormbase.org/species/c_elegans/variation/WBVar00089790#026-45-3
[9] Kullyev, A., Dempsey, C., Miller, S., Kuan, C., Hapiak, V., Komuniecki, R., ... Sze, J. (2010). A Genetic Survey of Fluoxetine Action on Synaptic Transmission in Caenorhabditis elegans. Genetics, 186(3), 929-941. Retrieved March 27, 2015, from http://www.genetics.org/content/186/3/929
[10] Hukema, R., Rademakers, S., & Jansen, G. (2008). Gustatory plasticity in C. elegans involves integration of negative cues and NaCl taste mediated by serotonin, dopamine, and glutamate. Learning & Memory, 15, 829-836. Retrieved March 27, 2015, from http://learnmem.cshlp.org/content/15/11/829
[11] Mohr, S., Smith, J., Shamu, C., Neumuller, R., & Perrimon, N. (2014). RNAi screening comes of age: Improved techniques and complementary approaches. Nat Rev Mol Cell Biol, 15(9), 591-600. Retrieved March 27, 2015, from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4204798/
[12] Waugh, T., Horstick, E., Hur, J., Jackson, S., Davidson, A., Li, X., & Dowling, J. (2014). Fluoxetine prevents dystrophic changes in a zebrafish model of Duchenne muscular dystrophy. Hum. Mol. Genet., 23(17), 4651-4662. Retrieved March 27, 2015, from http://hmg.oxfordjournals.org.ezproxy.library.wisc.edu/content/23/17/4651.full
Pictures from top to bottom:
http://topnews.com.sg/content/21392-parkinsons-disease-studied-help-zebrafish-scientists
http://www.vulcantermite.com/pest-profiles/the-house-mouse/
http://www.xatakaciencia.com/biologia/la-mosca-de-la-fruta-y-nuestro-sueno
http://akciospotencial.blog.hu/2007/10/03/nobel_dijas_fergecske
IMSR: http://www.findmice.org/index
FlyBase: http://flybase.org/
Bristle Screen Database: https://bristlescreen.imba.oeaw.ac.at/
WormBase: http://www.wormbase.org/#01-23-6
CGC Genetics: http://www.cgcgenetics.com/cgc/en/main-en.html
[1] ModelOrganisms - Home. (2012, January 1). Retrieved March 26, 2015, from http://www.modelorganisms.org/
[2] ZFIN Morpholino: MO1-slc6a4a. (n.d.). Retrieved March 26, 2015, from http://zfin.org/action/marker/view/ZDB-MRPHLNO-141031-1
[3] Waugh, T., Horstick, E., Hur, J., Jackson, S., Davidson, A., Li, X., & Dowling, J. (2014). Fluoxetine prevents dystrophic changes in a zebrafish model of Duchenne muscular dystrophy. Hum. Mol. Gen., 23(17), 4651-4662. Retrieved March 26, 2015, from http://zfin.org/cgi-bin/webdriver?MIval=aa-pubview2.apg&OID=ZDB-PUB-140513-91
[4] Holmes, A., Lit, Q., Murphy, D.L., Gold, E., Crawley, J.N. (2003). Abnormal anxiety-related behavior in serotonin transporter null mutant mice: the influence of genetic background. Genes Brain Behav., 2(6):365-80. Retrieved March 26, 2015, from http://www.ncbi.nlm.nih.gov/pubmed/14653308?dopt=Abstract
[5] Bengel, D., Murphy, D., Andrews, A., Wichems, C., Feltner, D., Heils, A., ... Lesch, K. (1998). Altered brain serotonin homeostasis and locomotor insensitivity to 3, 4-methylenedioxymethamphetamine ("Ecstasy") in serotonin transporter-deficient mice. Mol Pharmacol., 53(4), 648-655. Retrieved March 26, 2015, from http://molpharm.aspetjournals.org/cgi/pmidlookup?view=long&pmid=9547354
[6] JAX Mice Database - 008355 B6.129(Cg)-Slc6a4/J. (2009, November 24). Retrieved March 26, 2015, from http://jaxmice.jax.org/strain/008355.html
[7] EMMA strain search: B6.129-Slc6a4tm1Kpl/Cnrm. (n.d.). Retrieved March 26, 2015, from https://www.infrafrontier.eu/search?keyword=EM:05002
[8] N822 (variation). (n.d.). Retrieved March 27, 2015, from http://www.wormbase.org/species/c_elegans/variation/WBVar00089790#026-45-3
[9] Kullyev, A., Dempsey, C., Miller, S., Kuan, C., Hapiak, V., Komuniecki, R., ... Sze, J. (2010). A Genetic Survey of Fluoxetine Action on Synaptic Transmission in Caenorhabditis elegans. Genetics, 186(3), 929-941. Retrieved March 27, 2015, from http://www.genetics.org/content/186/3/929
[10] Hukema, R., Rademakers, S., & Jansen, G. (2008). Gustatory plasticity in C. elegans involves integration of negative cues and NaCl taste mediated by serotonin, dopamine, and glutamate. Learning & Memory, 15, 829-836. Retrieved March 27, 2015, from http://learnmem.cshlp.org/content/15/11/829
[11] Mohr, S., Smith, J., Shamu, C., Neumuller, R., & Perrimon, N. (2014). RNAi screening comes of age: Improved techniques and complementary approaches. Nat Rev Mol Cell Biol, 15(9), 591-600. Retrieved March 27, 2015, from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4204798/
[12] Waugh, T., Horstick, E., Hur, J., Jackson, S., Davidson, A., Li, X., & Dowling, J. (2014). Fluoxetine prevents dystrophic changes in a zebrafish model of Duchenne muscular dystrophy. Hum. Mol. Genet., 23(17), 4651-4662. Retrieved March 27, 2015, from http://hmg.oxfordjournals.org.ezproxy.library.wisc.edu/content/23/17/4651.full
Pictures from top to bottom:
http://topnews.com.sg/content/21392-parkinsons-disease-studied-help-zebrafish-scientists
http://www.vulcantermite.com/pest-profiles/the-house-mouse/
http://www.xatakaciencia.com/biologia/la-mosca-de-la-fruta-y-nuestro-sueno
http://akciospotencial.blog.hu/2007/10/03/nobel_dijas_fergecske