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

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Reverse genetics studies in Arabidopsis

How to set up a reverse genetics experiment with anArabidopsis thaliana mutantMining Phenotypes

1The 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 informationGeneral information: araport@jcvi.orgJason Miller, Grant Co-Principal Investigator, JCVI jmiller@jcvi.orgThis lesson was prepared by Andrea Cobb, Ph.D. (adcobb@fcps.edu) with the help of Margot Goldberg (mgoldberg1@pghboe.net)22PhenotypeA detectable traitExamples of detection:Visual (number of flower petals per flower)

Immunological (human blood type)

Biochemical (antibiotic resistant bacteria)3You might want to consider adding behavior to the list. Plants sense and respond to environmental information and this is often what we are looking for in the Experimental pair of pots in a PREP experiment. We change the information the plants are receiving, and then try to determine if the mutant plants response is different than that of the wild-type plants. This doesnt tell us if the cause is in the plants sensing capability or if its in the plants responding capability, but it helps us to narrow down a specific role for the gene of study.3Genotype DNA sequence of a gene

Gene variants

Homozygous or heterozygous

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Phenotype GenotypeTraditional 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

5Re: known phenotype Not sure if youll talk about some of the large mutagenesis screenings that are now famous forward genetics stories. One of my favorites is the one done on fruit flies by Christiane Nsslein-Vollhard and Eric Wieschaus (https://www.youtube.com/watch?v=7pSch5AoM24). Its a great modern science story.5Reverse genetics Scientists alter the genotype (mutate) Detect resulting change in phenotype Learn something about the genes function

Genotype Phenotypehttps://www.youtube.com/watch?v=5gyl_ODuZdYThis links to a short video with Dr. Gillaspy describing her reverse genetics research.6Arent 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

7Reference from conversation with Christ Town, JCVI 7

8Variations in phenotype might result from:Environmental interactionsEpigenetic regulationCopy number variants Multiple gene interactionsPleiotropy-one gene has multiple effectsMutationsknockouts, knock-downs and adding genes (transgenic)99Loss of function mutants-knockoutsPoint 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

1010Mutants with reduced gene expression-knock downWorks well when homozygous knockouts prove lethal

RNAi mediated

From Wikipedia11CRES-T a transcription activator of interest can be fused to a peptide or protein that converts it into a transcriptional repressor, which dominantly suppresses the expression of its target genes even in the presence of the redundant transcription factor.

11Transgenic plantsHow adding a gene may affect a plants ability to respond to the environment

https://www.youtube.com/watch?v=EW-3hgE6XpALinks to a PREP video1212How do scientists screen phenotypes? Visually-abnormal morphology, growth rate, color, flowering, fertility, etc.

Biochemicallyalterations in basic cell processes (replication, protein synthesis, etc.)

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

http://www.illuminatedcell.com/autophagosomes.html13Phenotype screening, continuedDevelopmental 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

14Phenotype screening, continuedCo-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:10.1038/415123ahttp://www.learner.org/courses/biology/textbook/proteo/index.html provides helpful information on functional proteomics.15http://www.learner.org/courses/biology/support/redbioact02.pdfProvides an excellent paper model of how scientists measure protein-protein interactions. 15Challenges to determining phenotype-genotype relationshipsMutants usually have more than one induced mutationwhich 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 *16How 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.17

Researching treatment ideas may increase the likelihood that you will generate meaningful data.18Many students seek to set up experiments to treat plants without understanding the physiology and cell biology behind such potential treatments. 18

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

19From 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 Windows50 years of Arabidopsis Research21Access other detailed information about Arabidopsis genes in ThaleMine

22Mine ThaleMineEnter the gene name into the search boxSelect the Gene category and you will see the gene information page

23Use the information and links on the gene page to decide:What might I measure about the plant?During what part of the plants 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?

24Gene 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 processesMolecular functionCellular componentThese may help you think about what to measure and how to measure 25Gene 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 expression26A closer look at gene expressionWhat 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)

28Proteins 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 genes 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

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http://www.ukhairdressers.com/romez.jpg

http://newborns.stanford.edu/PhotoGallery/Teeth3.html

https://en.wikipedia.org/wiki/Andr%C3%A9_the_GiantToo 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. 30

www.scq.ubc.ca/wp-content/cDNAarray.gifMicroarray experiments How do scientists measure gene expression?31

https://www.systembio.com/lncrna-research/disease-long-non-coding-rna/how-it-worksQuantitative Reverse-Transcriptase PCRHow do scientists measure gene expression?32How do scientists measure gene expression?

http://www.nature.com/ni/journal/v13/n9/fig_tab/ni.2407_F1.htmlNextGen RNA-sequencing