Biology 2202: Genetics Laboratory
Laboratory 1: Transgenes and GloFish
Laboratory handout
Introduction
by Samantha Lindeman and Jennifer Liang
Department of Biology, University of Minnesota-Duluth
Zebrafish have been used as a model organism to study genes that control development for about
thirty years. More recently, they have also been used as a model system for identifying disease genes and
even as sensors of environmental contaminants. Zebrafish were chosen as a genetic model system for many
reasons. (1) They are small, and so you can keep many genetic strains in a small space. (2) They produce
many progeny-a single pair of adults can lay 100-200 eggs in a single spawning, and they spawn about once
a week. You will see today how having lots of progeny aids in genetic analysis. (3) They are vertebrates,
and so the genetic control of their development and of genetic diseases is similar to humans. (4) Zebrafish
are much cheaper than mice, the other vertebrate genetic organism. (5) Zebrafish embryos are translucent,
making it easy to identify mutants with abnormal development.
Today we are going to study Mendelian genetics and statistical analysis using a zebrafish strain
called GloFish. GloFish are the first genetically modified, transgenic pet, and were first sold to consumers
in 2004 (http://www.glofish.com/). A transgenic organism is one that has a foreign gene introduced into its
genome. The gene that has been introduced is called a transgene. GloFish transgenic zebrafish were
developed initially by Dr. Gong Zhiyuan at the National University of Singapore to act as sensors of water
pollution (http://www.glofish.com/).
The transgene introduced into GloFish is composed of two parts. The first part is the promoter and
the second part is the coding sequence for a reporter protein. A promoter controls where and when a gene
will be expressed. The GloFish promoter is the muscle-specific mylz2 promoter (Figure 1). A reporter
protein is used to mark cells. The GloFish transgene encodes fluorescent reporter proteins that are originally
from jellyfish or coral or other organisms that have endogenous fluorescence. There are now many strains
of GloFish, expressing Green Fluorescent Protein (GFP), Red Fluorescent Protein (RFP), Yellow
Fluorescent Protein (YFP), Blue Fluorescent Protein (BFP), and Purple Fluorescent Protein (PFP)
(http://www.glofish.com/)(Vick et al., 2012). The mylz2 muscle promoter is so strong that the fish are
brightly colored even under normal white light. We call the transgenes in the Glofish GloGFP, etc.
(Lindemann et al., 2011).
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Laboratory Procedure (also see worksheet)
The goal today is to gather data and use statistical analysis to figure out the genetics of the fish that
we are examining. You will be looking at various groups (“clutches”) of zebrafish containing
wildtype, transgenic, and mutant fish. Based on your observations and data, you will generate a
hypothesis as to the type of genetics that are present, and test your hypothesis using a chi-square test.
You will also test part of your hypothesis by doing a cross between fish of your choice.
The fish that we are using today have combinations of three different genetic changes:
1. Glo transgenes, which produce bright body color under white light (Figure 1). Today, we have fish
carrying YFP, RFP, BFP, GFP, PFP.
2. The golden mutation, which causes the naturally dark stripes of zebrafish to be much lighter or nonexistent (Lamason et al., 2005)(Figure 2).
3. The long fin mutation, which causes the fish to have long fins (van Eeden et al., 1996)(Figure 3).
Figure 1. Multicolored GloFish
http://www.glofish.com
A
B
Figure 2. golden mutant phenotype
A. Phenotype of wildtype, striped zebrafish
B. Phenotype of zebrafish carrying golden
mutation
Figure adapted from Lamason et al., 2005.
A
Figure 3. long fin mutant phenotype
A. Phenotype of a wildtype female adult. Note the dorsal and
ventral lobes of the tail fin are of equal size.
B. Phenotype of a long fin female adult. Note that the ventral lobe
of the tail fin is longer than the dorsal lobe, and the whole tail fin is
longer relativeAto the length of the body.
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These mutant genes/transgenes could have four different types of inheritance:
1. Dominant – allele or phenotype that is expressed in either the homozygous or the heterozygous
state (AA or Aa).
2. Recessive – allele or phenotype that is expressed in only the homozygous state (aa).
3. Codominant- the phenotype of both alleles are present (such as when you have one black feathers
allele and one white feathers allele, the bird has both black and white feathers).
4. Incomplete (partial) dominance- condition that results when the phenotype of one allele in not
completely dominant over another allele and the heterozygous form exhibits a phenotype that show
traits of both homozygous forms of the alleles (such as when you have white feather allele and one
black feather allele, and the bird has grey feathers).
By examining the progeny of the different crosses we have available, you are going to match each mutant
gene/transgene with the correct type of Inheritance. For example, you might find that your data supports
the hypothesis that the long fin mutation is codominant. It is important to note that none of the genes we are
studying in this lab are linked (on the same chromosome)-we will work on linkage later in the semester.
Zebrafish nomenclature (same as fruit flies)
You will be expected to use these conventions in your laboratory reports:
gene names are lower case italics: The long fin gene is mutated in fish with long fins.
mutant names are lower case italics: Today, we are using long fin mutants.
mutant and gene names have a three letter abbreviation
transgenes have all different kind of rules-we are going to use capitalized, non-italics to differentiate them
from mutant genes.
GloFish transgenes are GloXFP, with the X corresponding to the color (G for GFP, etc.)
Glo- is used to indicate absence of a Glo transgene (since transgenes are not a normal part of the genome,
there is no such thing as a wildtype allele. Normal, grey fish have no transgenes)
long fin is lof
golden is gol
protein names are capitalized, non-italic: Green Fluorescent Protein
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Example:
GloPFP / Glo- ; + / gol X GloPFP / Glo- ; + / gol
GloPFP/Glo- = hemizygous at the PFP allele
+/gol = heterozygous at the golden allele
Chi-square test
The chi-square test is an important analytical tool frequently used to analyze Mendelian genetics.
Chi-squared is a statistical test used to evaluate how closely observed values match expected values (Pierce,
2008). For example, for a recessive mutant gene, a cross between two fish heterozygous for the mutant gene
is expected to yield one-quarter of the progeny with the mutant phenotype. If your observed values are 70
progeny with a wildtype (normal) phenotype and 30 progeny with the mutant phenotype, the chi square test
will tell you if the hypothesis of the mutant gene being recessive is correct or not correct. Ultimately, the
chi-square test yields a probability (P) that tells you the chance that your observed results and your
observed results are significantly different from one another.
It is very important that you understand what the P value means. If your P-value = 1, this means that
there is a 100% chance that your observed values and your expected values are not significantly different
from one another. Scientists usually put a threshold for deciding the validity of a hypothesis at P=0.05. If
P>0.05, the expected and observed values are not significantly different from one another, and the
hypothesis is supported; if P<0.05, the expected and observed values are significantly different, and the
hypothesis is not supported.
.
References:
Pray (2008) Recombinant DNA technology and transgenic animals. Nature Education 1(1).
Lamason, R., Mohideen, A., Mest, J., & Wong, A. (2005). SLC24A5, a Putative Cation Exchanger, Affects
Pigmentation in Zebrafish and Humans. Science 310, 1782-1785.
van Eeden et al. (1996) Genetic analysis of fin formation in the zebrafish, Danio rerio.
Development 123:255-62.
GloFish website (http://www.glofish.com)
Pierce, B. (2008). Genetics: A Conceptual Approach. New York, NY: W.H Freeman and Company.
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Vick, B. M., A. Pollak, C. Welsh, J. O. Liang. 2012. Learning the Scientific Method Using GloFish.
Zebrafish 9:226-41.
Lindemann, S.*, J. Senkler*, L. Auchter, J. O. Liang. 2011. Using zebrafish to learn statistical analysis and
Mendelian genetics. Zebrafish 8:41-55. *co-first authors
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