Planaria regeneration
GOALS of the EXPERIMENT
To understand regeneration and stem cell in the context of cell specification and
development.
To learn how change in gene transcription can be studied (Differential gene
expression, AKA DGE)
To practice navigating databases on NCBI and retrieving gene-related
information
Planaria general Information
Your animals are energetically motile, and this will be a disadvantage when you start to
make your cuts. The animal can be slowed significantly by cooling it. One method is to
transfer the animal to a glass slide that is sitting on ice. Since you will have to do all
your cuts under a dissecting microscope, realize that the light will be heating up your
specimen, counteracting the effects of the ice, so keep the light at a distance if possible.
Cuts can be made with microknives, two microneedles pulled across one another in a
scissor action or a scapel/razor blade. If you use a single microknife or blade to cut, you
will find it convenient to have a needle or probe in the other hand to steady the animal
during the cutting.
A thin slice of a specified width can be made by using two razor blades. Tape the two
blades together with a spacer such as a small piece of cardboard positioned between
the two blades to give the desired width for the cut. This device also makes the two cuts
simultaneously rather than sequentially, which is an important element in the
experiment.
A hole of a specified diameter can be punched out of the organism by using a Pasteur
pipette. Break off the bill of the pipette at the level that will give the bore size you want.
The unpolished end of the pipette is an excellent cutting device and can be used as you
would use a cork borer.
When the animal is stretched out, quickly make your cut. If you need to manipulate or
transfer pieces, use microneedles or a soft paintbrush. You may find that the cuts and
manipulations you make are not the ones you had initially planned. Don’t despair.
Record carefully what you did.
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Whatever cuts you make:
 record their exact positions using diagrams,
 save all your pieces
 number each piece individually recording in your notebook which cut it was
 You can also take a picture of the pieces after the cut with the Moticam.
Each piece can give you information about regeneration.
You will be keeping your regenerates over a period of time. Since regenerates move
and climb over partitions in a dish, each regenerate should be put into a separate small
petri dish. Be sure to label the dishes with your name, the datem and the number
assigned to each piece.
The water in your cultures must be changed frequently, every other day or so. You need
to agree with your partner when you will be coming. Be aware of times when the lab is
used for classes other than Developmental Biology.
You will need to be careful not to throw out the regenerate with the bathwater. Using a
pipette to remove the old water is the safest method. Also, remember that chlorinated
tap water is toxic. Use only the provided bottle spring water for your cultures.
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Schedule
Week 1:
Introduction to Planaria as a model system for regeneration and stem cell.
PowerPoint : regeneration
Discussion:
RNA expression analysis techniques. Available PowerPoint how to study RNA
You will work in groups of two and rotate through 3 activities related to planaria
regeneration. On the class list identify your group number with your name
STATION 1: Dissections:
1) Get familiar with the organism
http://www.nwabr.org/education/pdfs/STEM_CELL_PDF/StemCellCurriculumPOWERP
OINTS/PlanariaPP.ppt#284,19,Slide 19
2) Agree on the cuts you will make. You need at least 3 head and 3 tail pieces. Take
pictures of your animals before and after cutting.
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Examples of possible cuts
1)
2)
3)
Take notes related to the procedure (like “I butcher that one”, or this was a very
small piece…).
Label the plates with your group number and your sample number.
Put each piece into separate wells with SPRING water. Make sure to keep track
of which pieces go in which plate.
STATION 2: Pipetting and setting up a RT-PCR reaction
This is a mock exercise to practice pipetting and understand RT-PCR protocol
STATION 3: Bioinformatics
Practice navigating different databases and finding basic information using a mouse
gene from the list below and obtain the following:
1) Image of the gene structure
2) Number of exons, and possible alternative splice products
3) Size of gene, transcript(s) and encoded protein(s)
4) Predicted protein function and GO terms
5) Protein domain(s) if any
Note: the exercise is done with a mouse gene as most planarian genes are not yet
available on NCBI
Gene list:
Group1
Pax6
Group2
Wnt
Group3
Shh
Group4
MyoD
Group5
Patched
Group6
FGF8
Group7
c-myc
Group8
β-catenin
This is due at the end of lab posted in Dropbox in OAKS. Use the formatted answer
sheet posted in Dropbox.
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HOMEWORK:
After lab review the provided PowerPoints
Keep coming to change water. Discard any dead animals (record the information).
Record progress of regeneration by taking notes and pictures, and at the decided times
freeze the animals (record the information). Place animal pieces in a 1.5 mL Eppendorf
tube, label with name and time of freezing, and place at -200C.
Write introduction to lab report (see separate file for instructions).
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Week 2:
Presentation on how to study RNA expression:
What are the different methods with their advantages and disadvantages
(including microarray and RNAseq)
What is a transcriptome
What are the approaches to study differential gene expression across multiple
samples
STATION 1
Check your animals one last time. Record and take pictures. Freese any remaining
samples.
STATION 2
Puzzle exercise. You will have to take turn, but you can alternate with the other 2
stations. This is to illustrate the pipeline for RNAseq data assembly and matching to the
genome.
Materials for preparing the puzzles
STATION 3
Mining the planarian transcriptome profiling.
Use the following link to access the GEO file:
http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE56178
1) Familiarizing with the project and GEO datasets
The first page provides general information on the project
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Make sure to expand the section to see all the samples. The 24 samples include
triplicates of the following 8 conditions
Head regeneration samples at 0, 1,2, and 3 days after amputation
Tail regeneration samples at 0, 1,2, and 3 days after amputation
When you click on any of the GSMxxxx link, a page specific to that sample will open
and provide more protocol information
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2) Formulating a hypothesis
Discuss with your partner and decide which two conditions you will compare. Prepare a
short HYPOTHESIS related to your choice. For example I will compare head and tail
regeneration at 72 hours, hypothesizing that at this point, genes involved in the
regeneratio of end-specific structures will be activated, like the eye spot in the head.
Record in your notebook then proceed to the next step, which is the analysis itself.
3) Using GEO2R
You will use a software package GEO2R which provides a STATISTICAL evaluation of
the differences inRNA expression between the two chosen condition for each gene of
the planarian genome.
You will need to be back on the first page of the GEO dataset and expand the sample
list. At the bottom of the list you will see
Click on the link.
Because you need triplicates for a valid statistical analysis, each condition has 3
samples. The software will ask you to define two groups. Each group consists of the 3
files for each condition.
After you click on “Define group”, enter a name for your first condition. In my example I
will have “Head 72h”. click the ENTER key. You should see something like this:
By scrolling down, select the corresponding 3 files (on a PC you can select all 3 at once
by holding the SHIFT key, sorry Mac people, I am not sure). Then click on the group
name in the small window. The following should appear where the 3 samples have been
assigned to the group:
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Do the same for the second condition, e.g. tail 72 hours
Scroll to the bottom of the page and ckick on “Top 250”. GEO2R will calculate the
normalized difference in RNA amount for each gene and report the 250 most significant
genes.
This will generate a table that you can download and open with Excel.
HOMEWORK
Collect and label all your images from the planaria dissection.
Write result sections on the cuts you did and the extent of regeneration (see separate
file for instructions).
Review PowerPoints
Week 3:
Discussion on data normalization and meaning of FPKM and other statistical values.
STATION 1
Each group will choose two genes from the GEO2R table and perform the identification
using the Planaria genome browser found at the SmedGD website:
http://smedgd.neuro.utah.edu/index.html. Follow te instructions posted in the
accompanying PowerPoint on OAKS.
For each of your 2 genes include
1) Image of the gene structure
2) Number of exons
3) Size of gene, transcript and encoded protein
4) Predicted protein function from orthologs
5) Protein domain(s) if any
Download the gene sequence. Produce a WORD file with the exons and introns
Identified and the splice sites labeled.
Create the corresponding cDNA file and perform the translation.
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This is due at the end of lab in DropBox. Use the formatted answer sheet posted in
Dropbox.
HOMEWORK
Write result section on the two chosen genes
Review all PowerPoints
In PMC find a recent publication (last 3 years) dealing with planaria regeneration. Download.
Read it and prepare a short summary for the class next week (group work).
Week 4:
I will have done the RNA extractions on selected pieces and order the gene-specififc
primers
Set up the RT-PCR reactions with the primers designed for one of the gene you chose.
Paper presentation/dicsussion
HOMEWORK
Review all PowerPoints
Week 5:
1) Gel electrophoresis for RT-PCR
2) Wrap up and discussion
Report due in 2 weeks. I will post the gel images ASAP on OAKS
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