In this lesson, students will mine data from Araport.org to design and propose a reverse genetics experiment using a known Arabidopsis mutant. They will select a treatment to reveal phenotypic dfifferences between wild type and mutant Arabidopsis. Student handout and teacher resources are available at www.Araport.org, teacher resources. Suitable for grades 9-12 or first year undergraduate students.

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

2. 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: araport@jcvi.org
Jason Miller, Grant Co-Principal Investigator, JCVI
jmiller@jcvi.org
This lesson was prepared by Andrea Cobb, Ph.D.
(adcobb@fcps.edu)
with the help of Margot Goldberg (mgoldberg1@pghboe.net)
2

3. Phenotype
• A detectable trait
• Examples of detection:
– Visual (number of flower petals per flower)
– Immunological (human blood type)
– Biochemical (antibiotic resistant bacteria)
3

4. Genotype
DNA sequence of a gene
Gene variants
Homozygous or heterozygous
4

5. Phenotype  Genotype
Traditional “forward” genetics
known phenotype  work out genotype
https://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 5

6. “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_ODu
ZdY
This links to a short video with Dr. Gillaspy
describing her reverse genetics research.
6

7. 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
7

8. 8

9. Variations in phenotype might result
from:
Environmental interactions
Epigenetic regulation
Copy number variants
Multiple gene interactions
Pleiotropy-one gene has multiple effects
Mutations—knockouts, knock-downs
and adding genes (transgenic)
9

10. Loss of function mutants-”knockouts”
Point mutations –chemicals /EMS
Insertions/deletions (indels)
T-insertions
CRISPR-Cas9
Must be back-crossed to
reduce heterozygosity but sometimes that
results in a lethal combination
https://www.youtube.com/watch?v=QEbVpj7Ebw
U shows how plant scientists can change
Arabidopsis genes 10

11. Mutants with reduced gene
expression-knock down
Works well when homozygous knockouts prove
lethal
RNAi mediated
From Wikipedia
11

12. Transgenic plants
How adding a gene may affect a plant’s ability to
respond to the environment
https://www.youtube.com/watch?v=EW-
3hgE6XpA
Links to a PREP video
12

13. 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 13

14. 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
14

15. Phenotype screening, continued
Co-expression data Protein-protein
interactions
From the following article:
Proteomics: Protein complexes take the bait
Anuj Kumar and Michael Snyder
Nature 415, 123-124(10 January 2002)
doi:10.1038/415123a
http://www.learner.org/courses/biology/textb
ook/proteo/index.html provides helpful
information on functional proteomics. 15

16. 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/235171
22 * 16

17. 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 information
Mutant and wild type Arabidopsis are available at
ABRC.org and some commercial providers.
17

18. Researching treatment ideas may increase the
likelihood that you will generate meaningful data.18

19. A great place to begin desgining your reverse
genetics experiment
is at www.araport.org
19

20. From the Tools pull-down menu, select
50 years of Arabidopsis Research
20

21. 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
citation
Add 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
21

22. Access other detailed information
about Arabidopsis genes in ThaleMine
22

23. Mine ThaleMine
• Enter the gene
name into the
search box
• Select the Gene
category and you
will see the gene
information page
23

24. 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?
24

25. 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
25

26. 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_JTgk
Links to a short overview of gene expression
26

27. A closer look at gene expression
What is gene expression?
How do scientists measure gene expression?
27

28. Genes can be:
• Turned on (expressed)
• Turned off (not expressed)
• Turned up (increased expression)
• Turned down (decreased expression)
28

29. 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

30. http://www.ukhairdressers.com/romez.jpg
http://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.
30

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

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

33. How do scientists measure gene expression?
http://www.nature.com/ni/journal/v13/n9/fig_tab/ni.2407_F1.html
NextGen RNA-
sequencing
33

34. 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. 34

35. Examine the Developmental map
This will help
you think
about when
to collect data
during the
plant’s life
cycle.
35

36. • http://www.prepproject.org/
Matches the developmental stage to the
number of days after planting.
36
On which days after planting Arabidopsis seeds might
you need to collect data?

37. 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).
37

38. 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