Download Drosophila-Mega-Review

Flies Mega-Review
Basics
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4 chromosomes
o Chromosome I is the sex chromosome
 Females: XX
 Males: XY (XO is sterile male)
 No meiotic recombination in males (doesn’t matter which chromosome)
 Y has few genes (heterochromatic) so generally, if you put a transgene
on the 1st chromosome it would be on the X chromosome
o Chromosomes II, III are large autosomes
o Chromosome IV is a tiny autosome – mostly heterochromatin
o 99% of genes are on chromosomes I, II, and III. So, we will ignore chromosome IV.
Some code:
o Separate chromosomes with semicolons
 Remember, we can ignore the 4th chromosome
 I ; II ; III
 If a genotype is always +/+, you can ignore it
 Instead of +/+ ; UAS-GFP/+ ; Ubi-Gal4/+
 You can write UAS-GFP/+ ; Ubi-Gal4/+
o Separate (trans)genes on the same chromosome with commas
 UAS-GFP,Ubi-Gal4/+ ; Gal80ts/+
o
Single male:
 Usually used in a cross to isolate a certain genotype (in screens, to isolate
different mutations in mutagenized males)
o
Multiple males:
 Usually used for a cross if you’ve already isolated your genotype/mutant allele
o
Single female:
 Use with caution (might already be inseminated by males from their stock
bottle!)
o
Multiple females:
 Use with caution for same reason as above
o
Single virgin female:
 Use if trying to isolate genotype on the female side – for purposes of Pat’s
lectures this isn’t usually done since mutagenesis is done in males
o
Multiple virgin females:
 For purposes of the screens described in Pat’s lectures, you almost always use
multiple virgin females
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Balancers
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Important Characteristics:
o Suppresses recombination
o Includes dominant marker
o Homozygous lethal
How to write out balancers: “BALANCER, Dominant marker”
o Ex: Second chromosome balancer with a Curly marker:
 SM5, Cyo
st
o 1 chromosome balancers: FM1, FM2, FM3…etc
o 2nd chromosome balancers: SM1, SM2, SM3…etc
o 3rd chromosome balancers: TM1, TM2, TM3…etc
o Or, for purposes of the final, you can make up something:
 “I am using a first chromosome balancer named Purp that gives flies purple
eyes.”
You can also use double balancers; for example, TM1,Sb/TM2,Ser THIS IS VIABLE
If a mutatation is homozygous lethal, you can use balancers to keep a stock:
lethal = homozygous lethal mutation
lethal/TM3,Sb X
lethal/TM3,Sb 
lethal/lethal (dead)
TM3,Sb/TM3,Sb (dead)
Lethal/TM3,Sb (alive, stubble)
The Gal4 System
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Uses: to control transgene expression (reporter, RNAi, dominant negative) in a spatially and/or
temporally specific way
Components:
o Driver-Gal4: you can control where Gal4 protein is expressed using a tissue-specific
driver
o UAS-Transgene: Gal4 binds to the UAS promoter region to activate transgene
expression. Therefore, the transgene is expressed wherever Gal4 expression is driven.
 You can also use this to drive any transgene (RNAi to knock down gene
expression, dominant negative) in certain tissues/at certain times
o Gal80: For optional temporal control (protein inhibits Gal4 activation of UAS)
 tub-Gal80 - tubulin driver constitutively expresses Gal80
 tub-Gal80ts (Gal80 protein active at 18 deg Celsius, inactive at 29 deg Celsius)
Flp/frt system
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Uses: to induce clonal populations of negatively-marked cells (that can also be homozygous
mutant for a gene that is homozygous lethal to the organism when mutant)
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Components:
o Frt sites: inserted at the base of the chromosome arm at which you want flippaseinduced recombination to occur
o Flippase (flp): driven by heat-shock (hs-flp) or a Gal4/UAS system (UAS-flp)
o Ubi-Reporter (or UAS-Reporter if you want clonal induction to be tissue-specific)
o Driver-Gal4 (if you want clonal induction to be tissue-specific)
o Recessive mutant allele: if you want your negatively marked cell to be mutant - put on
the arm at which you want flippase-induced recombination to occur
MARCM system
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Uses: to induce clonal populations of positively-marked cells (that can also be homozygous
mutant for a gene that is homozygous lethal to the organism when mutant)
Components
o UAS/Gal4/Gal80 system
o Frt sites
o hs-flp
o Recessive mutant allele: if you want your positively-marked cell to be mutant - put on
the arm at which you want flippase-induced recombination to occur
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Flp-out system
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Uses:
o
Caveat for using flp/frt or MARCM system: only half of the cells are marked/become
homozygous mutant because mitotic recombination needed to induce clones
 Flp-out system doesn’t rely on mitotic recombination so has better chance of
marking all/most cells (greater clonal induction)
Components:
o Frt sites
 Flanking a STOP codon that disrupts expression of a transgene so that when
flipped out, transgene will be expressed
 Flanking a Gal80 construct that is inhibiting a Gal4 system, so that when Gal80 is
flipped out, Gal4-induced expression will begin
o flippase: (either hs or UAS-induced)
Practice problem: Mosaic analysis and crossing scheme to get desired genotypes
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