How to set up a reverse genetics experiment with an Arabidopsis thaliana mutant Mining Phenotypes 1.

How to set up a reverse genetics experiment with anArabidopsis thaliana mutant

Mining Phenotypes

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The Arabidopsis Information Portal is funded by a grant from the National Science Foundation (#DBI-1262414)

and co-funded by a grant from the Biotechnology and Biological Sciences Research Council (BB/L027151/1).

These lessons were developed during the summer of 2015 as education outreach for the www.Araport.org portal in conjunction

with the J. Craig Venter Institute, Rockville, MD, 20850, USA. We appreciate the lesson review and edits by David Lally at the

Partnership for Research and Education with Plants. Contact information

General information: [email protected] Miller, Grant Co-Principal Investigator, JCVI

[email protected] lesson was prepared by Andrea Cobb, Ph.D. ([email protected])

with the help of Margot Goldberg ([email protected])2

Phenotype

• A detectable trait• Examples of detection:– Visual (number of flower petals per flower)

– Immunological (human blood type)

– Biochemical (antibiotic resistant bacteria)

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Genotype DNA sequence of a gene

Gene variants

Homozygous or heterozygous 4

Phenotype Genotype

Traditional “forward” geneticsknown phenotype work out genotypehttps://blogs.brandeis.edu/flyonthewall/translational-findings-how-frui t-fly-research-has-already-contributed-to-human-health

find and sequence gene

http://cubocube.com/dashboard.php?a=1181&b=1260&c=103

http://www.sciencedirect.com/science/article/pii/S0005273699000644

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“Reverse” genetics Scientists alter the genotype (mutate) Detect resulting change in phenotype Learn something about the gene’s function

Genotype Phenotype

https://www.youtube.com/watch?v=5gyl_ODuZdYThis links to a short video with Dr. Gillaspy describing her reverse genetics research.

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Aren’t all Arabidopsis thaliana phenotypes known?

>13% of all Arabidopsis genes coding for proteins have a completely unknown function domains of unknown function

>30% of the Arabidopsis proteome is poorly characterized

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Variations in phenotype might result from:

Environmental interactionsEpigenetic regulationCopy number variants Multiple gene interactionsPleiotropy-one gene has multiple effectsMutations—knockouts, knock-downs

and adding genes (transgenic)

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Loss of function mutants-”knockouts”

Point mutations –chemicals /EMSInsertions/deletions (indels)T-insertionsCRISPR-Cas9 Must be back-crossed to reduce heterozygosity but sometimes that results in a lethal combination

https://www.youtube.com/watch?v=QEbVpj7EbwU shows how plant scientists can change Arabidopsis genes

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Mutants with reduced gene expression-knock down

Works well when homozygous knockouts prove lethal

RNAi mediated

From Wikipedia

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Transgenic plants

How adding a gene may affect a plant’s ability to respond to the environment

https://www.youtube.com/watch?v=EW-3hgE6XpALinks to a PREP video

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How do scientists screen phenotypes?

Visually-abnormal morphology, growth rate, color, flowering, fertility, etc.

Biochemically—alterations in basic cell processes (replication, protein synthesis, etc.)

Microscopically (use fluorescent tags to see overexpression) and image analysis

http://www.illuminatedcell.com/autophagosomes.html

Phenotype screening, continued

Developmental Metabolic or stress- dependent changes

Chemical genetic screens • http://abrcoutreach.osu.edu/educational-kits

http://web.stanford.edu/group/gcep/cgi-bin/gcep-research/all/lignin-management-optimizing-yield-and-composition-in-lignin-modified-plants/

http://journal.frontiersin.org/article/10.3389/fpls.2012.00004/full

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Phenotype screening, continued

Co-expression data Protein-protein interactions

From the following article:Proteomics: Protein complexes take the baitAnuj Kumar and Michael SnyderNature 415, 123-124(10 January 2002)doi:10.1038/415123a

http://www.learner.org/courses/biology/textbook/proteo/index.html provides helpful information on functional proteomics.

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Challenges to determining phenotype-genotype relationships

Mutants usually have more than one induced mutation—which mutation is crucial to effect?

Genetic redundancy in gene families-not all copies in the gene will be mutated, so normal phenotype may persist.

High throughput screening methods needed.http://www.ncbi.nlm.nih.gov/pubmed/23517122 *

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How does a scientist begin a stress or chemical screening experiment to explore phenotype?

https://www.youtube.com/watch?v=foHiKrlY9Qc is a PREP video which shows how!

Obtain the mutant and its genotype informationMutant and wild type Arabidopsis are available at ABRC.org and some commercial providers.

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Researching treatment ideas may increase the likelihood that you will generate meaningful data.

A great place to begin desgining your reverse genetics experiment is at www.araport.org

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From the Tools pull-down menu, select 50 years of Arabidopsis Research

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Enter the name of the gene into the search box.Scroll to see the related publications If you select one with a great number of citations, you can see the citation network.You can mouse over the network lines to see the citationAdd the publications of interest to Google Scholar

• App is written for Mac but will work for the most part on Windows

50 years of Arabidopsis Research

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Access other detailed information about Arabidopsis genes in ThaleMine

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Mine ThaleMine• Enter the gene

name into the search box

• Select the Gene category and you will see the gene information page

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Use the information and links on the gene page to decide:

What might I measure about the plant?During what part of the plant’s life cycle should I take measurements?How often should I take measurements?What might the gene product do?How might I measure what the gene product is doing? What treatments might make sense?What part of the plant should I use?

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Gene ontology is a systematic way of recording descriptive info about what a gene does, its involvement in processes, and where it is expressed.

Record the GO terms assigned to the gene:• Biological processes• Molecular function• Cellular component• These may help you think about what to

measure and how to measure

Gene expression is:

-The process of making a gene product (protein or RNA)

-The most basic mechanism whereby genotype gives rise to phenotype

https://www.youtube.com/watch?v=OEWOZS_JTgkLinks to a short overview of gene expression

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A closer look at gene expression

What is gene expression?

How do scientists measure gene expression?

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Genes can be:

• Turned on (expressed)

• Turned off (not expressed)

• Turned up (increased expression)

• Turned down (decreased expression)

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Proteins are the machinery of the cells. Proteins do many different jobs for the cell. Genes store information about making those different proteins. When genes are “turned on” (or expressed), the information in the gene’s DNA is used to make RNA. The RNA is then processed and is used to make

proteins. It is important to make just the right amount of protein at just the right time and place!

Figure 7-5 Molecular Biology of the Cell (© Garland Science 2008 29

http://www.ukhairdressers.com/romez.jpghttp://newborns.stanford.edu/PhotoGallery/Teeth3.html

https://en.wikipedia.org/wiki/Andr%C3%A9_the_Giant

Too much gene expression (growth hormone)

Wrong timing of gene expression (natal teeth)

Wrong location for gene expression (trichosis)

Changes in gene expression often change phenotype.

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www.scq.ubc.ca/wp-content/cDNAarray.gif

Microarray experiments

How do scientists measure gene expression?

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https://www.systembio.com/lncrna-research/disease-long-non-coding-rna/how-it-works

Quantitative Reverse-

Transcriptase PCR

How do scientists measure gene expression?

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How do scientists measure gene expression?

http://www.nature.com/ni/journal/v13/n9/fig_tab/ni.2407_F1.html

NextGen RNA-sequencing

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Examine Expression

• First look at the Tissue specific gene expression.

• This will guide you about what part of the plant to sample or observe..

• Look at the legend to see maximum expression.

• White indicates that data is unavailable or was not tested for that tissue. http://

www.hhmi.org/biointeractive/how-analyze-dna-microarray-data links to a lesson on microarrays used to generate this data.

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Examine the Developmental map

This will help you think about when to collect data during the plant’s life cycle.

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• http://www.prepproject.org/Matches the developmental stage to the number of days after planting.

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On which days after planting Arabidopsis seeds might you need to collect data?

Look at the other Expression pull-down menus

They may give you ideas about how the environment influences gene expression. You might want to select or modify one of the treatments for your stressor (or not).

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Proposal Ideas?

Work in groups of 4-6 to review and add to your notes.Brainstorm from your Araport notes and literature searches:What are possible functions for the gene product? .What phenotypic changes might be expected?How might we change the environment to elicit those phenotypic changes?How and when might we measure those phenotypic changes? 38