Ease of doing genetic experiments makes Drosophila an excellent model system to study development and disease. The reasons for ease in setting up genetic crosses and its subsequent interpretation of results can be attributed to many efficient genetic tools, balancer chromosomes, innumerable genetic markers with clear visible phenotype, mutants for many genes and less number of chromosomes.
The Drosophila genome of 180MB is distributed among 4 chromosomes, however majority of 13600 odd genes are present on first three chromosomes. The tiny 4th chromosomes has very few genes on it.
Hence only first 3 chromosomes are used for most of the genetic crosses, which makes it rather simple to manage and track when compared to 40 chromosomes of mouse or any other genetic model.
Handling or tracking fewer chromosomes is really advantageous in many aspects, however it gets sometimes difficult to incorporate three or more transgenes or mutants in one fly stock for genetic crosses.
As all required transgenes or mutants of interest are needed to be accommodated on just three chromosomes and sometimes two available transgenes are incorporated on the same chromosome.
Since we cannot have required transgenes or mutants on homologous chromosome as they tend to segregate from each during meiosis / gamete formation and this defeats our purpose of having both transgenes in F1 progeny.
This issue can be solved by recombining the transgenes or mutants present on homologous chromosomes, so that they end on one same sister chromatid. ( For more details on Homologous chromosomes and related terminology please refer)
Working example:
We will try to understand this better by taking two examples : If one wants to knock down a gene using a UAS- RNAi line in the background of a mutant of interest or perform a large scale RNAi screen to decipher genetic suppressors or enhancers of a gene of interest, it is important pay attention to few aspects.
To perform this genetic screen we need – A tissue specific or Ubiquitous GAL4 driver, Mutant allele of gene of interest and it would be useful to incorporate GAL80ts to overcome any undesired embryonic lethality which might be occur from knocking down of some gene using specific RNAi line.
Success of Genetic screens:
The success of any large scale genetic screen depends on proper planning in designing the crosses, generate appropriate stocks to have high percentage of individuals with all desired fly constructs / mutants in F1 generation and simple scorable phenotype (lethality, phenotype in eye etc) to analyze if the candidate gene from the screen interacts genetically with our gene of interest.
Designing genetic cross and generating fly stocks for the screen:
The basic idea behind the screen is to look for genetic interactors of the gene of Interest. So to screen many UAS-RNAi in quicker time with maximum percentage of individuals with all desired reagents ( GAL4, UAS-RNAi, GAL80ts and mutant) in F1 progeny, we need to generate a fly stock with GAL4, mutant and GAL80ts .
Theoretically it is possible to have all three components on three different chromosomes but it is rather difficult to generate a fly stock in that manner, hence not advisable.
To simplify matters we can recombine either the GAL4 with GAL80ts or Mutant allele with GAL4 or GAL80ts on to same sister chromatid, depending on the chromosome location of these fly reagents.
If The GAL4 driver and GAL80ts are on second chromosome then we can recombine them first and then make a stock of newly generated GAL4-GAL80ts recombinant with mutant present on third chromosome. Other possibility will be pick GAL80ts on third chromosome and recombine with mutant allele of interest.
Later GAL4 driver can be combined with new GAL80ts-Mutant allele recombinant to generate a stock to cross with various UAS-RNAi lines for large scale genetic screen.
We will look into details of both the scenarios.
Methodology- Recombination:
Recombination of GAL4 driver with GAL80ts : Suppose we are using act5c-GAL4 and TubpGAL80ts for recombination , both on second chromosome.
First we need to cross 10 virgin females and 7 males from either of the stock . Since the chromosomes involved for recombination are autosome and not the X chromosome, where X chromosome from the male parent is passed on to only female embryo and Y chromosome is contributed to males.
If X chromosome is involved in a genetic cross it is always advised to collect virgin females from the fly stock with X chromosome insertion or mutation, so that in next generation both males and females can receive the X chromosomal contents equally ( if insertion or mutation is not lethal in hemizygous condition).
The number of virgins and males can also vary but 10 females to 7 males is good enough number to get recombinants
After the cross in the next generation we need to collect around 10-20 act5C-GAL4 / TubpGAL80ts virgin females.
It is important to collect females at this point as recombination is absent in Drosophila males.
If we manage to collect 20 females of act5C-GAL4 / TubpGAL80ts then can be crossed to double balancer fly stock involving second and third chromosome, in two sets of 10 females to 7 males from double balancer stock.
In the next generation we need to carefully pick individual flies ( both males and virgin females) with eye color darker than the eye color of parental flies and cross to double balancer flies ( single mating crosses) .
One should be careful in comparing eye colors and it is important to compare eye color from female in F2 generation to females from F0 ( parent fly stocks ) and not with males of both parents.
In Drosophila , males generally have stronger eye pigment than females in a particular fly stock.
The reason behind selecting stronger eye color fly in F2 as a possible recombinant is based on the logic that the eye color in a transgenic fly comes from the mini white gene cloned in transformation vector used for injections.
So the darker eye color implies presence of two transgenic constructs in a single fly and might be a possible recombinant of act5C-GAL4 -TubpGAL80ts.
Hint: One can observe the eye color of F1 females very closely for get some help in selecting darker eye color recombinant. Since F1 flies contain both the transgenes, so a possible recombinant will be having a eye color of similar intensity.
However it is important to know that in presence of a balancer chromosome ( which is the case when we select recombinants in F2 ) the pigment intensity slightly reduces , whereas F1 females lacks balancers).
These recombinants should be balanced on both second and third chromosomes using double balancer stock ( pin / Cyo ; TM2 / TM6B ).
The stocks generated from Individual flies should be maintained separately and treated as different recombinants, however it is important to confirm the presence of both the transgenes by PCR or genetically by crossing to UAS-GFP at 18C and 29C separately.
If both act5C-GAL4 – TubpGAL80ts are present in the new stocks then we should not see any GFP expression in the cross maintained at 18C and green fluorescence should appear ubiquitously due to presence of act5C-GAL4 driver and inability of GAL80ts to repress GAL4 at 29C.
If both these criteria at 18C and 29C are fulfilled then we can treat that particular stock as a authentic act5C–GAL4 – TubpGAL80ts recombinant and can be used to generate final stock for the large scale genetic screen with mutant of gene of interest on third chromosome, which can be eventually crossed to different UAS-RNAi lines for checking suppressors or enhancers of our gene of interest.
This method of selecting recombinants based on eye color intensity will work only when eye color of parental geneotypes is anything other than dark red ( as in wild type Canton S flies). Even if one parent has dark red eye color then it is better to employ PCR method for screening recombinants as genetic method require to screen many possible recombinants stocks.
Recombination of mutant allele with GAL80ts :
In this section we will see how to recombine a transgene (GAL80ts) and a mutant allele. Here the eye color method will not work as mutant allele ( generated by CRISPR or P-element mutagenesis) usually doesn’t have any eye color or even if they exhibit some eye color , it cannot be used for final screening as it doesn’t come from any mini white construct of transgenes.
The first cross involves both female virgins and males from either of the parents and then virgin females are collected from F1 generation. They are crossed to males from double balancer stock and the individual flies with eye colour are selected for further crosses to generate possible recombinant stocks. The presence of eye color establishes presence of the transgene and this comes from TubpGAL80ts stock. To confirm successful recombination of mutant allele with TubpGAL80ts we need to confirm by PCR and further sequencing of gene of interest for possible presence of mutation.
If the mutant allele is homozygous lethal then it gets rather simple to confirm the possible recombinant by just checking for the presence or absence of marker from the third chromosome balancer in the individual recombinant stocks generated from single F2 flies. If mutant allele is present in the new stocks then we will see only balanced flies for third chromosome ( TubpGAL80ts – mutant allele / TM6B ) and not even a single fly without third chromosome balancer ( TubpGAL80ts – mutant allele / TubpGAL80ts – mutant allele). The confirmed TubpGAL80ts-mutant allele will be clubbed with act5c-GAL4 on second chromosome to generate final stock for the large scale genetic screen , which can be amplified in big bottles or vials to be crossed with different UAS-RNAi lines for checking genetic interactions of our gene of interest.
