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Introduction
Introduction
Welcome to the DarkJedi Project
Are you a student or teacher interested in hands-on genetics and genome editing? The Orange Maker Project offers an exciting opportunity to learn about Drosophila genetics, transgenesis, the GAL4/UAS system, Mendelian inheritance, somatic mosaicism, and CRISPR/Cas9 technology – all while contributing to real scientific research!
This project, developed by undergraduate students at the University of Minnesota, allows participants to create new variants of fruit fly lines with orange eyes. These "DarkJedi" lines are valuable tools for the Drosophila research community, making genetic crosses and selections easier.
Through a series of simple experiments, you'll use CRISPR/Cas9 to modify the eye color of red-eyed GAL4 fly lines, turning them various shades of orange. Along the way, you'll gain practical experience with:
Fruit fly handling and crosses
Basic molecular biology concepts
Cutting-edge genome editing techniques
Scientific observation and documentation
No specialized equipment is required, except for a confocal microscope for advanced imaging (which is optional).
Whether you're an undergraduate, high school student, or science educator, this step-by-step guide will walk you through the entire process. Join us in this educational adventure that bridges classroom learning with real-world scientific contribution!
Ready to start your journey into the world of Drosophila genetics? Let's begin!
Basic Knowledge
Basic Knowledge
How to select flies having two transgenes
Why do we need DarkJedi lines?
How to make DarkJedi lines (Genome editing: What is the CRISPR/Cas9 system?)
Drosophila Culture
Drosophila Culture
Drosophila Workers Unite! A laboratory manual for working with Drosophila By Michele Markstein
Fly stereoscope: Amscope SF2TRA or SM-1 series or Similar
Genome editing: What is the CRISPR/Cas9 system?
Genome editing: What is the CRISPR/Cas9 system?
Reading materials
-Molecular biology at the cutting edge: A review on CRISPR/CAS9 gene editing for undergraduatesWhy do we need DarkJedi lines?
Why do we need DarkJedi lines?
Step by Step Instruction
Step by Step Instruction
Generating your own Orange maker
From the Orange Maker (founder line provided by the Kim lab)
Step 1: Obtain the Necessary Fly Stocks
Step 1: Obtain the Necessary Fly Stocks
Orange Maker (founder line): y[1] M{w[+mC]=nos-Cas9.P}ZH-2A w[*]; attP40{U6.2-w-ex3-2}/CyO
Note: Ensure that the Orange Maker stock is healthy and well-maintained. Regularly check the stock for any abnormalities.
Target GAL4 Line: (e.g., OK371-GAL4 or elav[C155]-GAL4)
OK371-GAL4: Commonly used for targeting motor neurons.
elav[C155]-GAL4: Used for pan-neuronal expression.
Other GAL4 lines that have red eyes in heterozygotes.
Note: Confirm the expression patterns of your GAL4 lines before starting the crosses (How to).
Balancer Line: Bl/CyO (for OK371, Second chromosome) or TM3/TM6B (Third chromosome)
Note: Balancer lines are crucial for maintaining stocks and preventing recombination. Ensure these stocks are also healthy and verified.
Contact me to obtain fly lines
When you receive the flies, transfer them to fresh food vials and allow them to recover.
Step 2: Set Up the Initial Cross
Step 2: Set Up the Initial Cross
Collect Virgin Females:
Virgin females must be collected within 8-10 hours of eclosion to ensure they have not mated.
Preparing virgin females: a. Clear all adult flies from the Orange Maker vial. b. Wait 8-10 hours (at 25°C) for new adults to emerge. c. Collect newly emerged females. They will be virgins if collected within this timeframe. d. Identify females: look for larger abdomen, dark tip, and no sex combs on front legs.
Sex comb method (Youtube: )
Tip: frequent collection intervals to ensure the virgins. Drosophila Workers Unite! A laboratory manual for working with Drosophila By Michele Markstein
Cross with Target GAL4 Line Males:
Set up crosses in standard vials with fly food.
Setting up the cross: a. In a new vial with fresh food, add 3-5 virgin Orange Maker females. b. Add 3-5 males from your target GAL4 line (OK371 or elav[C155]). c. Label the vial with the cross details and date.
Note: Ensure a good ratio of females to males (e.g., 10 virgin females to 5 males) for optimal mating success.
Maintain the Cross:
Keep the cross at 25°C.
Tip: Monitor the vials daily for fungal or bacterial contamination and replace food if necessary.
Step 3: Screen for Mosaic-Eyed Flies
Step 3: Screen for Mosaic-Eyed Flies
F1 Generation Screening:
Look for mosaic-eyed flies, which indicate successful Cas9-mediated cutting and subsequent homologous recombination.
Tip: Use a stereomicroscope to carefully examine eye color. Mosaic eyes will have patches of red and white.
Collect Appropriate Flies:
For OK371, collect mosaic-eyed males.
For elav[C155]-GAL4, collect mosaic-eyed females.
Note: Handle the collected flies gently to avoid stress and ensure they are transferred to fresh vials quickly.
Step 4: Cross Mosaic-Eyed Flies
Step 4: Cross Mosaic-Eyed Flies
For OK371:
Cross individual mosaic-eyed males with virgin Bl/CyO females.
Tip: Set up multiple crosses to increase the chances of obtaining successful offspring.
For elav[C155]-GAL4:
Cross individual mosaic-eyed females with w[1118] males.
Note: Label each cross vial carefully to keep track of parentage.
Step 5: Select Orange-Eyed Flies
Step 5: Select Orange-Eyed Flies
Next Generation Screening:
Look for flies with uniform orange eyes, indicating successful integration and expression of the transgene.
Tip: Use a good light source to differentiate between shades of eye color.
For OK371:
Collect orange-eyed males.
Cross these individually with virgin Bl/CyO females.
For elav[C155]-GAL4:
Collect orange-eyed males.
Cross these with virgin w[1118] females.
Step 6: Establish Stable Lines
Step 6: Establish Stable Lines
For OK371:
Collect virgin females and males with orange eyes and CyO.
Cross these to establish a stable OK371[DJ] line.
Note: Regularly check the progeny for the presence of the balancer and orange eyes.
For elav[C155]-GAL4:
Collect orange-eyed males and females.
Cross these to establish a stable line.
Select homozygous virgin females (identified by stronger orange eyes).
Cross these with orange-eyed males to establish the final elav[C155][DJ] line.
Tip: Maintain detailed records of the crosses and phenotypes.
Step 7: Verify GAL4 Expression
Step 7: Verify GAL4 Expression
Cross with UAS-GFP Line:
Use JFRC81-GFP or a similar reporter line to verify GAL4 activity.
Note: Ensure the UAS-GFP line is healthy and has a robust GFP expression.
Check GFP Expression Patterns:
Examine salivary glands and ventral ganglions without dissection using a fluorescence stereoscope.
For detailed analysis, dissect larval brains and image using a confocal microscope.
Tip: Compare GFP expression patterns with the original GAL4 lines to ensure similar functionality and specificity.
Step 8: Maintain and Document Your Lines
Step 8: Maintain and Document Your Lines
Keep Detailed Records:
Document eye color intensity, any observed variations, and other phenotypic characteristics.
Assign each line a unique DarkJedi (DJ) designation (e.g., OK371[DJ1], OK371[DJ2], etc.).
Tip: Use a database or spreadsheet to keep track of all lines and their details.
Maintain Lines:
Regularly transfer lines to fresh food vials to prevent overcrowding and ensure healthy populations.
Note: Periodically verify the genotypes and phenotypes to confirm line integrity.
Additional Considerations
Additional Considerations
Validation:
Further validation through genomic sequencing may be required to confirm the presence and integrity of the transgene.
Tip: Backcross to w[1118] to remove any unintended modifications.
Troubleshooting:
If mosaicism is not observed,
Safety and Ethics:
Follow all relevant guidelines for working with genetically modified organisms (link).
Ensure proper containment and disposal of transgenic flies and materials (link).
Additional details are coming!
Further analysis
Further analysis
Sequencing Strategy for DarkJedi lines
Sex-linked lethality
p{GawB} map, sequence, target site
Currently available DarkJedi and related lines (Please request by e-mail)
Currently available DarkJedi and related lines (Please request by e-mail)
Orange Maker: Genotype y[1] M{w[+mC]=nos-Cas9.P}ZH-2A w[*];attP40{U6.2-w-ex3-2}/CyO (white eyes, the mini-white gene, w[+mC], was inactivated in this line)
OK371DJ: Genotype w[1118]; P{w[+mW.hs]=GawB}VGlut[OK371]DJ from #1 to #6
elavC155DJ-GAL4: Genotype: P{w[+mW.hs]=GawB}elav[C155]DJ from #1 to #4 P{w[+mW.hs]=GawB}e
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