Biology 2202: Genetics LaboratoryLaboratory 1: Transgenes and GloFish

Laboratory handout

Introductionby Samantha Lindeman and Jennifer LiangDepartment 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 non-

existent (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 2. golden mutant phenotypeA. Phenotype of wildtype, striped zebrafishB. Phenotype of zebrafish carrying golden mutation Figure adapted from Lamason et al., 2005.

Figure 1. Multicolored GloFishhttp://www.glofish.com

Figure 3. long fin mutant phenotypeA. 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 relative to the length of the body.A

AA

B

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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.

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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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