Lecture #9 - Drosophilla 1 Flashcards
Why is Drosophilla a good model
- Many genes and biological processes are conserved in flies (Ex. 75% of human disease causing genes have homologs in Drosophila meaning we can swap human homologs)
- Inexpensive
- Easy to maintain
- Small
- Short life cycle
- Produce many offspring (females lay 100 eggs per day)
- Simple genome (Have four chromosomes = designing genetic crosses is simple)
- Array of sophisticated genetic tools are available
Answer - E
Can store dead embypres in the freezer BUt they would not be alive after
Drosophila genome
15k protein-coding genes
Higher gene density than humans
Drosophilla Life cycle
Have a short life span (40-50 days)
Four stages in Drosophila development - egg + larva + pupa + adult
Life cycle - Egg is fertilized –> after 24 hours it will undergo embryonic development and hatch into a larva –> The larva subsequently undergoes three molting phases called instars (instar 1 –> 2 –> 3) –> Next, the pupa enclosed in a pupal case undergoes metamorphosis and the body structure is rearranged and the adult structure
- Instar 1 and 2 = last one day vs. the third instar = last two days
- Adult flies begin mating about ten hours after eclosion (emergence from pupal case)
- Life cycle is dependent in temperature
What type of females are used in genetic crosses
Need to use female virgins when setting up a genetic cross to ensure parentage of the progeny
- Important because females can store sperm from males
Drosophila – Tissues + development + brain functions
There is a lot of conservation of development in drosophila and humans –> A lot of the foundational understanding of development came from drosophilla
Example - Can stain for different features of the embyroe
- Top image – patterning genes that set up the body plan (early embryo)
- 2nd image – Trachea
- 3rd image – Muscles (Figured out how cell-cell fusion works by studying flies )
- 4th image – Nervous system in flies
- Image in middle – Larave neuromuscular junction (study in flies)
- Right image – dopamine neurons in brain in adult (study brain using flies)
Circadian rythms + flies
Circadian rhythms = discovered in flies –> work in fruit fly led to nobel prize in understanding the central clock that regulates fly and human circadium rhythms (molecularly conserved)
- Molecular conservation = led to understanding of basic biology
Polytene chromosomes
Polytene chromosmes = Giant chromosomes formed by multiple rounds of DNA replication without cell division (DNA is replicated 1000s of times)
Polytene chromosomes are found in certain cells in flies
Use - Early fly geneticists used polytene chromsomes to decipher the structure and arrangement of the genome
- PolyT chrosomes = how poeple made maps of chromosomes
Drosophilal chrosome cytological maps
Able to look at PolyTene chromosomes by squashing the cells and flattening the nucleus content (including the chromsomes)
- Looking at PolyT chromosome = gave people link between chromsomes and where genes are in position along the chromosome
Bottom part of image – see PolyTene chromosome
- See dark and light areas –> shows areas with more or less DNA condensation (The pattern is reproducible across flies)
- Image shows bands 20-40 on second chromosome left arm
- Have pictures for all chromosomes in flies
TOP picture = shows the bands on all arms of the chromosome (X has 0-20 ; 2 has 21-60 ; 3 has 61-100 )
ANSWER – 3R (because 94 is between 81 and 100 – look at last slide)
Can do FISH for all genes and they will map to band on chromosome (green signal)
- Upper micrograph = have 94e1 THEN do FISH and see a fluorescent band
Sexing Flies
Male (XY):
1. Smaller
2. Black and rounded abdomen
3. External genitalia
4. Have sex combs (patches of dark bristles on the foreleg for mating)
Female (XX):
1. Larger than males (BUT could have mutations that change body size)
2. Internal genitalia (no Sex combs)
3. Striped and pointed abdomen
Y does NOT determine maleness –> INSTEAD the lack of a second X chromosome determines maleness
Drosophila chromosomes
Drosophila have 4 pairs of chromosomes
- Chromosome 1 = the sex chromosome
- Chromosomes 2-4 = autosomes
Chromosome 4 – dot chromosomes (very small ; 2% of genome)
Chromosome 2 and 3 = have their centromere positioned in the center which seperates each chromosome into a right arm and a left arm (Each arm carries about 20% of the fly’s genes)
Drosophilla genome
132 million base pairs (1/20 the size of humans)
15k gene
Gene density is much higher than the human genome
Karyotype - Shows have 2 pairs of chromsome 2 + shows chrosmome 3 i+ shows the X chrosmome in orange + shows chromsome 4 as two dots
Do we care about chromosome 4
Often don’t care about chromsome 4 in crosses because has so few genes
- Often don’t include when writting genotypes of flies
BUT Chromosme 4 does HAVE essential genes
- Hard to resrecah things on chromosome 4 because it has a lot of heterochromatin
Genetic Nomenclature
Wild type = wt or +
Gene name and genetic elements is Italicized (Curly)
- Genes in flies are given the name of the mutant phenotype
Gene symbole is itallized (abreaviate name of gene) - Ex. Cy
Dominant mutations are capitalized (Ex. Dr or Cy)
Recessive mutations are written in lowercase (Ex. vg or hh )
Allele of gene uses a super script (allele name is written as a super script)
- Example - W^a –> allele of white gives rise to a pale orange eye color (allele = apricot)
- Example 2 – hh^8 –> 8th allele of hh gene (allele name is superscript)
Writting a genotype
Overall - the genotype of each chromosome is indicated separately
Chromosomes are separated by semicolons
- Chr 1 ; Chr 2 ; Chr 3
Each homologous chromosomes are separated by a line
- Maternal chr 1 / paternal Chr 1 ; Maternal chr 2 / paternal Chr 2 ; Maternal chr 3 / paternal Chr 3
Different alelles on the same chromosme are seperarated by commas
- +/+ ; Allele 1, Allele 2/ + ; +/+ (Allele 1 and 2 both on chromosome 2)
The sex chromosomes can be written in multiple ways
- Female = +/+ or x/x
- Male = +/ weird symbol or x/y
Answer – C –> has 2 domiannt mutations on chromsome 2 and a recessive mutation on chromsome 3 and it is male
ANSWER – B
Answer – C
w- = means have 2 X chromosomes (w-/w-)
Chr 2 = lowercase = recessive
Ch 3 = uppercase = Dominant
Answer E (Know it is E because hh is on the second chromsome (based on A) AND NOT D – because gig is on the 3rd chrosome (Based on A
w is mutant
- w- is an allele of w mutant
Shortcuts to write fly genotypes
Might not write out all of the chromosomes (if one chromosome does not have any alleles of note on it it won’t be written)
If a pair of chromosomes are homozygous THEN only one chromosome will be shown
Sometimes alleles are not separated with a comma
Sometimes allele names are not written out
Example – Cross a male that is heterzygous for Cy (on 2nd chromosome) and Heterzygous for dr (on the 3rd chromsome) TO females that is homozygoyz for eb (recceisve mutation on the 3rd chromsome)
Each offspring receives one chromosome from each homologous pair from each parent
Because the female is homozygous for every chromosome, there is only one combination of maternally inherited chromosomes –> +; + ; eb
Male can give 8 possible combinations (Can give + or Y on chromsome 1 ; can give Cy or + on chromsome 2 ; can give Dr or + on chrosome 3)
END - There are eight possibilities for the genotypes of the offspring (because 1 possible from female and 8 possible form male)
Calculating offspring ratios
Maternal gametes – w or +
Paternal gametes – w or Y
SLIDES = shows possible offspring
Punnet square = shows you the distribution of gamete
ANSWER – ¼
How to assign sex to the parent genotypes
Need to consider many things when thinking about which parent genotype will be male or female in the cross:
1. The chromosomal location of genes within the parental genotypes
2. 2 - If there needs to be meiotic recombination to produce the desired F1 progeny
3. If one of the parent genotypes has a limited amount of flies
4. If you are going to mutagenize the parents
How to assign sex to the parent genotypes - Chromosomal location of genes within the parental genotypes
If the gene to be inherited by the F1 generation is on the X –> the parent with the X containing gene must be female
Example – IF you want progenicy with baltimore in genotypes
- When cross a fly that is homozygous for the gene Baltimore on the X chr and WT on the other chromosomes to a fly that is homozygous for the gene Maryland on the 2nd chr but WT on the other chromosomes –> the homozygous Baltimore fly must be the female to generate progeny with 1 copy of Baltimore in their genotype (see genotype on slide)
- WHY - If we were to make the homozygous Baltimore fly male, then only the the female progeny (NOT the male progeny) will have 1 copy of Baltimore in their genotype
How to assign sex to the parent genotypes - Chromosomal location of genes within the parental genotypes - If there needs to be meiotic recombination to produce the desired F1 progeny
If there needs to be meiotic recombination to produce the desired F1 progeny
Only females can do meiotic recombination = you must use females parents
BOTH males and females can do mitotic recombination.
How to assign sex to the parent genotypes - Chromosomal location of genes within the parental genotypes - If one of the parent genotypes has a limited amount of flies
If one of the parent genotypes has a limited amount of flies –> should make that the male parent
WHY - males do not have to be virgins prior to mating –> means it is easier to use males when there is a limited number of flies with that genotype
How to assign sex to the parent genotypes - Chromosomal location of genes within the parental genotypes - If you are going to mutagenize the parents
If you are going to mutagenize the parents you should mutagenize males
WHY – because males generate more gametes than females –> means that there is higher potentoal for more uniquley mutagenized progeny
Fowards genetics in Drosophilla
Fowards genetics – Go from phenotype to genotype
Uses:
1. Random point mutatiions (EMS or radiations)
2. Random Insertions (P-elements)
3. Enhancer or supressor screens
4. Deficicey kits
Reverse genetics in Drosophila
Reverse Genetics – Go from genotype to pheotype
Uses:
1. Gene Knockdown (RNAi)
2. Knockins/deletions (CRIPSR)
3. Overexpression (GAL-4-UAS)
4. Cell types specific expression (GAL-4-UAS and clonal analysis)
Generating Mutations in Flies
- Chemical mutagens (EMS) –> Makes Point mutations
- Pro – random + saturation + different types of muatnts
- Con – Slow gene identifications + large scale - Ionizing radiation (X-ray and gamma rays) –> Makes chromosome rearangments
- Pro – Random + gene deletions + duplication
- Con – Slow gene identification + no real saturatiion + inefficnet - Transposons –> Makes DNA insertiions (mostly hypomorphic in UTRs)
- Pro – fast gene identification + flexible scale
- Con – Non-random (hot spot) + no saturation - Homologous Recombination and CRIPSr/cas9 –> Makes trageted mutogensis of specfic
- Pro - Very efficient
- Con – Currentley ineffcincet for screening
Foward genetic Screen
Foward genetic Screen = unbiased screening for mutations producing a phenotype of interest
Example Fowards Genetics Screen
Goal – Screen for genes on second chromosome essential for photoreceptor function
- Fundemental for studying RAS – all unraveled by studying fly eye
Example Fowards Genetics Screen - What method would you use to generate mutation
ANSWER – EMS – want point mutations (most common)
- NOT CRSIP because not efficinet
- NOT trasnpons because would get large deletions/rearngemnmts
Example Fowards Genetics Screen:
1. Will you mutagenize males or females
2. 2 – What Drosophilla genetic tool will allow you to establish a mutant stock
1 – Will you mutagenize males or females
- ANSWER – Mutagenize males because males make more gametes + recombination is only in females –> means the mutant male won’t recombine and move the mutant around on the chromsome
2 – What Drosophilla genetic tool will allow you to establish a mutant stock
- Answer – Balancers
Fowards genetic Screen process
- Mutagenize male with EMS –> generates mutations across all chromosomes
Issue when mutagenize with EMS
Issue – EMS will cause a recessive mutation that we can’t visualize when the fly is heterozygous (looks WT) –> how do we keep track of a mutaion that we can’t see
Solution - Uuse balancers to keep track of recessive mutations (allows you to follow recessive mutations)
- Because EMS is genreating recessive mutations that we can’t see in heteroygous fly = need to cross a balancer chromsome to recover the mutant
Drosophilla Bodies
Drosophilla have complex bodies and every morphical feature is fair game
Body structures and morpholigical features can exhibit chnages due to mutations
Drosophila Mutant Examples
Eye color is complex and ranges widley
Fly genetics can take advtange of this by linkning eye color to a gene (Ex. Link to GFP) –> NOW if eye color is present then that means GFP is also present
Image - shows mutations that cause chnages in eye shape
Rerousces for fly genes
The most popular public database is FlyBase (flybase.org)
Use - Use it to search for your gene of interest using the search bar
Example – serach for hedgehog (hh)
- Results on flybase show that hedgehog is a protein coding gene (it is a singlaing pathway ligand) ; located on the right arm of chromosome 3
- Website also tells us that - If we are interested in studying hedgehog further (Ex. perhaps knocking it down or overexpressing it) –> we can find 39 publicly available stocks
- Can also see Molcular fuction + Expression patterns + Alleles + Phenotypes resulting for LOF or a mutation of a gene
- Can get the DNA sequence of a gene by clicking on “get decorated FASTA”
Issue in fly crosses
Issue – How do you miantain a lethal mutaion in the heterozygous state
Example – Cross parents that each carry a recessive lethal mutations (on 2nd chromosome)
- Half of the progencey will be heterozygous for the mutatinos ; ¼ of progencey will be WT ; ¼ of the progeney will be homozygous for leathal mutations (¼ will die)
Solution - Balancer chromosomes
Balancer Chromosome Use
Overall prevent reccesive mutations from being lost over time + prevent recombination between homologous chrosomsomes
Use – Balancer chromosomes allow us to maintain a mutations (ex. lethal mutations) in the heterozygous state and trace a mutation through the generations
- Balancers are important IF the mutation is homozygous lethal
- Useful if flies with the mutations can’t be phenotyoically distiguished from the WT fly (Used to trace a mutation that cannot be phenotypically distinguished from a wildtype fly) –> because the flies carrying a balancer chrosome can be phenotypically distigushed from flies without a balancer chromosme
Nomenclature of balancers
Image – show shows how you write balancers + how you maintain in stock
In(2lR) = inversion of the 2nd chromsome
Cy = dominant marker
dp = recessive lethal (don’t see in heterozygous state)
pr^1cn^2 = recessive visible marker (don’t see in heterozygous state)
Use of Recessive visible marker
Recessive visible marker – used to show ressive lethal works in marker (because if recessivle lethal stops working then you would get homozygous and would be able to see the recesive visible marker)
Mainatining balancers
Overall - Balancers need to be kept in a heterozygous state = they are kept opposite of a difefrent dominant marker
Balancer chromsomes are miantained as stocks with a dominan viable lethal mutation (ex. Pin - maintain flies as Pin1/SM6c)
- When flies cross for a while they will keep the Pin1/SM6c genotype
- Dominant visible lethal mutation = visible phenotype is domiant but lethality is recessive
- Need PIN to stay heterozygous PIN/SM6c
Why does PIN maintain balancers in stock
PIN is dominant for visble phenotype BUT recesive for lethality (can’t have Pin/PIN) and can’t have SM6/SM6 = can only have Pin/SM6
END - PIN shows you the flies that are heterozygous for SM6c and Pin1
Features on balancer chromosome
Balancers have 3 features that distiguish them from a WT chromosome
- Dominant marker
- Recessive lethal mutations
- Inversions over the length of the chromosome to supress recombination
Dominant marker on Balancers
Use - Allows us to see which flies have the balancer
Dominant marker is NOT lethal
Example (in image) - curly and Pin
- Flies with a balancer that has the curly marker have curly wings OR Flies with a balancer containing Pin have shortened bristles that look like pins
Recessive lethal mutations on Balancers
Use - Have recessive lethal mutations so that flies can NEVER be homozygous for the balancer chromosomes AND they can never be homozygous for the mutant chromsome –> MEANS that the mutations of interst (which is on the chromosome that is being balancers) can be maintained in the heterozygous state
END - Flies have to be heterozygous balancer/chromosome with mutation (flies have to have the recessive mutation of interest and balancer chromosome)
- Means that the balancers can never outcomepete the mutation of interest (limites the propagation of chromosomes)
Inversions in Balancers
Use - Supress Recombination
- Inversion breakpoints occur at the same points where the recessive lethal mutations map
The inverted regions are largely WT except for their orientation –> means that with a balancer you are supply a nearly WT version of the chromosome = keeps the heterozygous healthy
How do the balancers suppress recombination - When a balancer and WT recombine = creates an acentric and dicentric chromosome –> Unhealthy –> leads to death = eliminates recombination (because cells would die if they had recombination)
WHY is it important that the inversion suppress recombination
IF you want to study a double mutant (double mutant exist as a heterozygous) –> during meiosis when recombination occurs you could end up with what you want (have no recombination and get m1 and m2 on the same chromsome) or can get recombination and have chromoeosme with just m1 or just m2
THEREFORE balancers can suppress this recombination due to heir inversion and maintain the double mutant that you want
How do balancers prevent you from losing mutants of interest
IF have parents that is m/+ X +/+ –> progeny will be m/+ or +/+
- IF these offspring flies mate THEN over time you will lose m mutations because m is homozygous lethal –> homozygous lethal mutatons usually give disadvantage even when heterozygous
IF have parents that have 2 homozygous lethal mutants (m1 and m2) - Cross m1, + / + , m2 X m1, + / + , m2
- Some of offspring is m1,+ / m1, + OR m2,+ / m2, + = dead
- 1/3 of offsrping ate m1,+ / +,m2 = heterozygous (alive = allows you to maintain the mutations)
- IN reality – female flies can recombine chromosmes = you won’t mainatin the two mutations on that chromsomes
- To solve issue of recombination = put inversions on chrosmomes = supress recombination –> allows you to make balance stock (Lose ability to do recombination = won’ be able to lose m1 or m2 = maintain mutations )
Balancer Notation
Balancers usually start with the letters F, S, or T –> indicates if they balance the first, second, or third chromosomes
Example - SM6b = second chromosome balancer and contains dominant markers for rough eyes and curly wings
- True
- True (Has domiannt marker)
- False (has recessive lethal)
- True
- Generally True
- False
Why can you use PIN in males
Can use Pin to balance a gene in males because they don’t do recombination (don’t need inversion to prevent recombination if there is no recombination BUT could not use in females)
- PIN = does not have inversion = it is NOT a balancer
- Need SM6c in females because it is a true balancer
Forward screen process:
Foward screen process: EMS males to make mutations across genome –> Different mutations are made –> Cross to balancer females (cross to virgin females of balancer stock) –> Single male cross –> sibling cross –> Anylyze homozygous phenotype
- When chromosomes are mutated –> each chromosmes will have a spectrum of mutations –> can have many alleles of a gene = each fly needed to be handled individually because different flies will ahve unique spectruum of mutatons + some chromosmes might not have mutations
Crossing mutagenized males to a balancer female
After EMS –> Cross mutagenized males to a balancer female
- Get a mix of genotypes and phenotypes
AFTER cross - Select for flies with SM6c (curly wings) –> mut1/SM6c or +/SM6c (select for flies that don’t have PIN)
- Because mutations are recessive – you would not be able to tell mut1/SM6c and +/SM6c apart (ALSO both would look the same as mut2/SM6C - could also not tell apart from +/SM6c)
Foward Screen - Single male crosses
Once have mut1/SM6c or +/SM6c or mut2/SM6c –> do single male crosses
Have to seperate out the males and cross each single male to many virgin balancers (Pin/SM6C)
- NOW we are just dealing with 1 mutation per cross
- Done so that you only have 1 mutation per fly (ex. only have mut1)
Foward Screen - Sibling cross
From EACH single male cross you select for progeny (both male and virgin females) that have SM6c but not Pin THEN do sibling cross
Once have SM6c flies = set up sibling cross = create a balanced stock
Foward Screen - Analyze the homozygous phenotype
From the F2 siblings you select the progeny that do NOT have SM6c = NOW have mut1/mut1 or mut2/mut2
- You can look if any of the mutants have the phenotype you are looking for
- Many of the homozygous mutant flies will not have the phenotype we want = this type of screen requires thousands of flies and hundreds of crosses
- Could screen for any phenotype
Example - Sequencing of the 2nd chromosome shows that a gene called eos is mutated in flies with defecteive Photorecptors (Mutant allele = eos^23 )
EMS results in mutations all over the genome –> how can you show that the mutations in eos are responsible for the defective PRs
To show that mutations in eos are respinsible for defective photoreceptors you do a phenotypic resuce
Phenotypic analysis – adults without SM6c are homozygous mutants –> look for phenotypic rescue in non-SM6c flies
How do you do a phenotypic rescue
- Establish transgenic line carrying heatshock inducible WT eos (eos+)
- Add transgene under induucible promoter to restore gene function
- Cross in image = shows how to do this
- Apply heat shock to induce transgene function
- Cross the transgene into your mutant background
- Look for phenotypic rescue in homozygous mutants after heatshock
Application - Can determine developmental time period when eos is needed by heat shocking at different times
2nd method for expressing transgenes
SAY you have a hard time rescuing eos using hs-eos+ since expressing eos+ throughout the body is bad and INSTEAD you want to express eos+ specifcially in photoreceptors
NOW you would use the tissue specifc expression of GAL4/UAS binary expression system (NOW you can express specifically in photo receptors)
GAL4-UAS - Overall
Use – Directed gene expression with spatial (in tissue/cell type) and temperal control (life stage)
System is adapted from yeast – creates specificty in the fly
Why might we want temperal and spatial control over the gene expression pattern
Control is good IF you want to study gene’s function in a specific tissue at a specific point in development BUT over expression or reduction of the gene in the whole organism may be lethal + don’t want to over complicate the experiment by altering gene expression in more tissues than desired
Solution – Use GAL-4-UAS to overexpress the gene of interest in the specific tissue you are interested in
Components of GAL4-UAS binary gene expression system
- GAL4
- UAS
GAL4
GAL4 = Transcription factor
Gene encoding GAL4 is inserted using P-elements downstream of enhancers –> determines where GAL4 will be transcribed
- Enhnacers can be tissue specifc = tissue specifc GAL4 expression
Example - Enhancer can only be expressed in the wings = creates a fly line that only expressed GAL4
UAS (Upstream activating sequence)
UAS = Enhancer than GAL4 binds to and affects expression of the downstream gene
- UAS is inserted into the fly genome
In a tissue where enhancer is active –> GAL4 is expressed THEN GAL4 binds to UAS –> Activates trancrtiption of the downstream gene (get expression of the gene in tissue specific manner)
Is GAL4 endogenous to flies
GAL4-UAS is NOT enodgenous to flies –> means a fly with ONLY UAS or ONLY GAL4 expression would be otherwise WT (won’t get expression of the downstream gene)
Important controls because if there is an effect in the flies with only GAL4 or only UAS transgene it would change your interpretation of the experiment
Answer - B
The gene downstream of the UAS is expressed only when/where the GAL4 is expressed, which is dictated by the enhancer element(s) located nearby the GAL4 insertion site
Answer - 4
Scenerio – have GAL4 that is in eos gene –> Cross GAL4 with UAS FP reproter
How would verify that the expression pattern you go relfects the real expression pattern of gene itself because using transgene to report the expression of genes but need to valdiate
ANSWER – Look for GFP expression and verify for anti-eos antbody (immunostain for protein) , FISH or other reproter in the same gene
Rescueing a mutant with GAL4-UAS
Can learn about mutations/KO with GAL4 UAS system in order to ask questions about resucing a phenotype
Example – Mutants (hopkin) –> cause a malformation of eyes
- GOAL – want to express the WT version of the gene in the eye in the mutant background
How to do this – make a fly line with the mutant allele and GAL4 (GAL4 would be under and enhnacer that is expressed ONLY in the eye) THEN you can idetify the desired progeny using a balancer chromosome
- CyO helps us follow the mutant alelle (mutant = maryland allele) because if have no curly wings then you have to be homozyous for mutant
- TM3 = helps us follow GAL4
Cross – males (have mutant hopkins with Cyo and eye-GAL4 balanced by TM3) to females that have WT hopkins donwstream of UAS (being balanced by TM6B)
Results from cross when rescueing a mutant with GAL4-UAS
AFTER make the cross = we will ONLY collect flies that do not have a balancer to ensure that the homozygous mutants have both GAL4 and UAS transgenes
WHY no balancer (Because on chromsome 2 need eye-GAl4 and UAS hopkins = the offspring with no balancer would have eye-GAl4 and UAS:Hopkins)
NOW we can see if overexpressing the WT hopkins resuces the mutant phenotype in the eye
Steps after rescue the mutant pheotype
AFTER Overexpress hopkins to see if it can resuce the mutant phenotype –> Might want to link structure to function and understand what part of the hopkins gene is doing a function
To Study – replace the UAS hopkins transgene with the UAS transgenes that are mutants of hopkins that ONLY disrupt parts of the gene
- Example - Looking at a kinase dead allele of the hopkins gene –> Now can ask – does expressing the kinase dead domain version of the gene rescue the mutant
- Instead of adding the full WT gene downstream of UAS you only add part of it to see which parts of the gene is needed
End - Using GAL4-UAS we can see how much and which part of the gene is needed for the rescue
ACTIVITY – phenotypic rescue of eos with GAL4/UAS
Slides = shows the cross you would preform to use GAL4/UAS + Slide shows progeny from cross
- Cross = mutant/balancer and eos-GAL4 X homozygous mutant and UAS/balancer
Using GAL4-UAS to find other genes in the pathway
Can use GAL4 system in screen to see if overexpression of other genes can rescue a KO of the gene of interest
- NOW see if overexpressing a different gene in the eye can resuce the mutant phenotype
Overall - CROSS the hopkins mutants with UAS upstream of different genes
Using GAL4-UAS to find other genes in the pathway - Experiment
Experiment – Start with fly that has gene of interst mutated that ALSO expressed GAL4 in the eye –> mate these flies to library of UAS flies –> Look for phenotype of interest in offspring
What type of gene can rescue the mutant phenotype
IF the mutant phenotype is rescued then you may have found a supressor gene of your gene of interest
CAN also find gens that make the phenotype worse than the KO alone
Other uses of GAL4
- Tissue specific knockdown using UAS-RNAi
- Temperal control of expression usning GAL80TS
Overall - Many RNAi and GAL80 fly transgenic lines are availible
Tissue specific knockdown using UAS-RNAi
Asking what is the phenotype of a fly that is missing the protein of interst ONLY 1 tissue (Ex. in the eye)
Here the shRNA is expressed under the UAS promoter –> expresion of shRNA decreases protein expression
Temperal control of expression using GAL80TS
Asking what is the phenotype of a fly that expresses the protein at a certain time
GAL80TS = temeprature sensative version of GAL4 inhibtor
- GAL80 = GAL4 inhibitor
Answer - B and C
A fly expressing only the GAL4 should not have any way of expressing a marker to indicate which cells it is expressed in. Therefore, you must cross your GAL4 flies to a UAS marker line, but not UAS:GFP RNAi as this would result in knockdown of GFP
RNAi would result in a Knockdown of GFP
Necessary
Necessary – Deleting your gene of interest creates mutant phenotype
Example – If you study fly development and find that flies without the eyeless gene have no eyes –> Shows that eyeless is necessary for eye development
- No eyeless gene = have no eyes –> eyeless gene is necessary for eyes
Sufficient
Sufficient – Rescuing mutant phenotype by introducing your gene
Example – Studying fly development and start with fly that has no eyes THEN express the gene eyeless –> flies NOW have eyes = shows that the gene is sufficient for development
- Add back in eyeless and get eyes = eyeless is sufficient
Neccessary Vs. Sufficient
GAl4-UAS is a good tool for studying necessity and sufficiency of gene of interest
Sometimes you can have one without the other = need to test both
