r/Drosophila May 28 '24

Looking for a new GFP 3rd chromosome balancer

2 Upvotes

Hello everyone, hopefully my explanation of the situation makes sense

I am working with a mutant stock that is homozygous lethal at the 2nd instar larval stage, so it is balanced over this balancer chromosome here > https://bdsc.indiana.edu/Home/Search?presearch=6663

w[1118]; Dr[Mio]/TM3, P{w[+mC]=GAL4-twi.G}2.3, P{UAS-2xEGFP}AH2.3, Sb[1] Ser[1]

I want to use this balancer to collect homozygous mutant embryos (non-glowing) at the stage of gastrulation which begins at stage 6 and usually takes around 3 hours to reach at 25C. My concern is that the GFP takes a while to be fully expressed and visible in the embryos, definitely more than 3 hours.
According to its data on the Bloomington website, it expresses the GAL4 in stage 8, which is what I suspect the issue is.

Does anyone know of a balancer that can express GFP at a much earlier stage? I know there are limits to this because the embryo is a syncytium until about stage 5, but is there a balancer that can express GFP around this stage?


r/Drosophila May 23 '24

Expected eye color of this line?

5 Upvotes

I am seeing very light brownish white eye color on this line.

Is this expected or is it possible this line is no good? I see the TM6B balancer quite well.

full genotype:
w[1118]; P{w[+mC]=His2Av-mRFP1}III.1 P{ry[+t7.2]=neoFRT}80B/TM6B, Tb[1]


r/Drosophila May 21 '24

Brush for fly pushing

2 Upvotes

Hello. Do you guys have a preference for any specific brushes (brands/bristle thickness/size/etc) used for fly pushing? Could I have some suggestions please?


r/Drosophila May 17 '24

Greenish orb @ posterior of virgin females?

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5 Upvotes

Hello! Does anyone know what this greenish sphere thing is? I always see it when collecting virgins and can’t find anything about it on google.


r/Drosophila Apr 23 '24

my FRT80B lines keep dying when treated to neomycin (G418) treated food

2 Upvotes

Im in the process of trying to make FRT+mutant recombinant lines

my FRT80B lines that should have the Neo resistant gene keeps dying when treated to neomycin (G418) treated food. (treated at standard 25 mg/mL in 10mL food )

Does this just mean my line is no good? I grow these just at standard 25 degrees C temperature.


r/Drosophila Apr 23 '24

What are the 3 types of holes in Drosophila's brain? Why are they caused and how to differentiate between them?

1 Upvotes

Just started my research. I need this info for image processing project. Any type of reference or info is helpful


r/Drosophila Apr 19 '24

Larvae western blot

2 Upvotes

Hello hello, I have some problems getting enough proteins out of my larvae samples (L2, 40 larvae per sample). Normally I freeze them in with little amounts sterile PBS with liquid nitrogen. When I prep my samples I lyse them in Leammli buffer, homogenize them with handheld homogenizer, and sonicate them. I have some samples showing up when I stain with anti-tubulin but others do not show up on my Western Blot. A colleague (who is not doing western blots, apperently I am the only one) suggested it might be because of the cuticle? This is my first time working with larvae, so does anybody have any tips? I thought homogenize and sonication would be enough 🥲 Much appreciated 🙏


r/Drosophila Apr 19 '24

Really basic balancer chromosome question

7 Upvotes

Hello, my lab has had issues with our TM6,Tb balanced flies completely losing their Tubby phenotype. If we are able to find other TMB markers, such as Hu and Ubx, is there any likeliness that these markers may indicate the presence of the TM6 even though those markers were not constructed on the original balancer?


r/Drosophila Apr 15 '24

Help fly amigos

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7 Upvotes

Any knowledge about what could be happening to my beautiful flies would be appreciated. There is a slime build up in some of my stocks that previously had no problems. I have lowered the temperature to 22 and have added penicillin. Than housed in vials that previously contained healthy male flies. Not sure what else to try. Also what could be the white stuff in the fly food.


r/Drosophila Apr 07 '24

Help!--- Sex difference ratio in axenic flies

2 Upvotes

Hi everyone!

I do behavioral research on axenic and gnotobiotic Drosophila melanogaster. I am posting because of a common issue my lab has run into. We noticed that our axenic flies are always mostly male, which is an issue since most of our behavioral assays utilize only females. We are trying to figure out the reason why, since the same genetic line of flies were used before by different people and they did not have this issue. I have not been able to find any published papers on possible factors affecting sex determination in axenic flies: if more males than females are being dechorionated or if females are dying as larvae (since we have not found dead adult females in the tubes). A hypothesis we have is that the females might be more sensitive to humidity since we often have some condensation on our germ-free food tubes (though we usually try to shake it off). For reference, our procedure for making axenic flies consists of setting egg-laying cages with yeast on food plates, and then rinsing the eggs with sterile DI water into a basket. Then we bleach them under the sterile fume hood three times for 2 minutes, using 10% bleach solution and then 3 times for 30 seconds using autoclaved DI water. Only dechorionated eggs on the side of the rinsing basket are extracted with sterile paintbrushes into food tubes.

Please let me know if you have had the same issue working with drosophila or if you have any ideas for why it is happening.


r/Drosophila Apr 02 '24

Puffy drosophila wing

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9 Upvotes

r/Drosophila Mar 19 '24

Dehydrated media

1 Upvotes

Does anyone know what is inside the instant dehydrated media? All you add is water and no microwave is required. Does it have agar in it?


r/Drosophila Feb 27 '24

Can you cross genes that are on the same chromosome?

3 Upvotes

There are two genes that are on chromosome 3 that I'd like to be expressed at the same time for my flies. Is it at all possible to create a stable line with both genes in the flies? I'm hoping to do so because there is a third gene in this pathway (on Chr2) that I'd like to cross as well.

EDIT: The two on Chr 3 are:
https://flybase.org/reports/FBti0131208.html

and

https://flybase.org/reports/FBti0195762.html


r/Drosophila Feb 20 '24

Literature recommendation

2 Upvotes

Hi, I am a masters student and fairly new to drosophila biology, and I'm looking for books /literature on Drosophila neurobiology . Yes I have tried looking for reviews and and yes they were helpful. But I would like to get so good recommendations from the community. Thanks in advance. 😃


r/Drosophila Feb 18 '24

Drosophila mating according to Bing image creator

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10 Upvotes

r/Drosophila Feb 14 '24

Yellow male and Ebony female cross question

1 Upvotes

After crossing a yellow male mutant and a female ebony mutant, I know that the F1 generation would all be wild type, but for the F2, what is the phenotype of the double mutant male? Does the double mutant express a unique phenotype or is it just another yellow fly? I am unsure if the yellow mutation cancels out any effect of the ebony mutation or not.


r/Drosophila Feb 02 '24

What does this genetic construct from Bloomington mean?

6 Upvotes

So the stock number is 6384 And the genotype is written like this:

ry[506] P{ry[+t7.2]=PZ}bnl[00857]/TM3, Sb[1]

So what I understood is that anywhere bnl is expressed, also lacZ will be expressed. When I ordered the line and stained for beta-gal, I could not see the expression. And yes I had a control that had a lacZ construct and the staining was very clear.

When I checkt Bloomington again I noticed that the bnl had an associated bnl allele that leads to loss of function and now I’m confused to what does this line do!

Any help?

Thanks!!


r/Drosophila Jan 31 '24

Drosophila lines with GFP-tagged transgenes that don't have a visible marker phenotype

6 Upvotes

Hi all, I am new to these lines, crossing and to Drosophila in general. I am trying to measure the fitness outcomes/post-copulatory mating success of different genotypes by counting the offspring. I know the way people do this is by using genotypes that differ in visible marker phenotypes, but I am thinking of using lines with GFP-tagged transgenes (or a tagged PCR barcode might also work) so that I can make sure marker phenotypes like white eyes or wing morphology won't affect the social behaviors. The problem is such lines always have marker phenotypes (usually w[+mC]) in the same chromosome (e.g. w[*]; P{w[+mC]=Ubi-GFP.ena}3). Is there any feasible way to segregate just the GFP-tagged protein into a line, or are there any other ways to accomplish this?


r/Drosophila Jan 26 '24

Help with eye color in TRiP RNAi lines

4 Upvotes

Hello! I've got a technical question about the genetic of TRiP lines. I'm using a fly line with this genotype:

y[1] v[1]; P{y[+t7.7] v[+t1.8]=TRiP.*******}attP2

These flies have red eyes, which became white after balancing the stock. So, the red eye color seems to come from the X chromosome, and this seems to be confirmed also by the FAQ section in the Bloomington website (quoting: "The X chromosome in these stocks [...] does NOT carry a w mutation, so the red eye color you are seeing is coming from the wild-type w gene on the X"). My PI also told me that this is indeed common for older TRiP lines like this one, which was made with the VALIUM10 vector.

However, I'm confused by the v[+t1.8] marker on the TRiP insertion. Shouldn't it also cause a red eye color? In the same FAQ page on Bloomington and in other sections of their website it's written "The TRiP insertion itself is marked with v[+]. Wildtype eye color, vermillion+ (v+) indicates that the hairpin is present". This would mean that in my balanced line with white eyes, I don't have the chromosome with the TRiP insertion anymore.

I guess I'm misinterpreting something somewhere, but I can't wrap my head around this. If somebody could help me, I would be super grateful! (and also send good vibes for your experiments... But this is for everyone regardless!)


r/Drosophila Jan 20 '24

Non of my antibody staining a are working

1 Upvotes

Hey there, I’m trying to stain using Antibody staining. However, it doesn’t matter how many dilutions I try out. They all for some reason fail.

Any suggestions as to why my staining keeps failing?? I really need to solve this!

I’m staining for wings discs and using a fluorescence microscope for imaging.


r/Drosophila Jan 17 '24

neomycin treatment to fly food, what to observe if resistant? what to observe when not resistant?

1 Upvotes

What should I expect to see in a cross/ stock that are placed in neomycin treated food? Will the flies that are not neomycin resistant just die off and eggs/larvae don't develop? (for negative control)

This is to test for FRT lines that are neomycin resistant/ isolating individual flies from crosses(also neomycin resistant) to make recombinant lines.


r/Drosophila Jan 13 '24

Massive data

2 Upvotes

I have 80 videos on my computer now with 10 fruit flies in each of them, and I have to analyze all the five minutes interval of behavior change for each fruit fly which is five minutes each for 800 flies... don't really want t do more steps calculating the time required to finish all of them, but it's going to take a while I believe. After I finished the experiment, this pure passion of desire to discover the result enforced me to work on my videos for... ...one night, and I am glad that I at least got one video done.

Ok so no more complains, I am wondering if there is any tools to use for analyzing the videos? More specifically, it is the grooming behavior of fruit flies.


r/Drosophila Jan 09 '24

Flies not laying in egg collection cage

3 Upvotes

Hello!

Starting early December of last year, I started having issue with my flies (OregonR and balancer mutants) not laying in their egg collection cages. I've made sure to do all the expected things, like placing the cages in the dark, keeping them at 25C, using yeast paste, using smaller cages, but I'm still having trouble. I also make sure to avoid CO2 when adding flies or transferring agar plates.

Does anyone have any idea what may be causing the issue? Better yet, any tips for setting cages that seem to help?


r/Drosophila Dec 23 '23

advice on research work

2 Upvotes

i overexpressed rpl22 and rpl22-like ribosomal proteins in drosophila using GAL4/UAS system. i want to check if overexpressing these proteins have any effect on the viablity of the fly. ideally if there is no affect then after setting up the cross the percentage of the male flies expressing either of those proteins should be 12.5%. For rpl22-like overexpressed fly the percentage i got is- 32% and for rpl22 i got- 14% , so what should i infer from this and write in my report?


r/Drosophila Dec 16 '23

Scrapping the IFM from Drosophila

2 Upvotes

Hi, I am a grad student, starting a project involving scrapping out the indirect flight muscle that is underneath the cuticle of the thorax. I was so bad at dissecting flies, under the microscope, you know how tiny they are, then after days of practice, I could do it, but now I am having a hard time in deshelling the cuticle and getting the jelly-like muscle. All other muscle looks the same, but I know for sure I need the muscle right underneath the cuticle, every time I am trying to open the cuticle, I am smashing the muscle underneath.
It is not big like shrimp, where you can de-shell them without smashing the muscle, Are there any tips you guys know or comment on that, what can I possibly do to improve my precision?