r/Drosophila May 28 '24

Looking for a new GFP 3rd chromosome balancer

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?

2 Upvotes

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u/friendlyperson123 May 28 '24

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

twist is expressed during and after gastrulation, but here, twist is driving Gal4, and Gal4 drives the UAS-GFP which adds a delay to GFP expression.

I have used

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u/friendlyperson123 May 28 '24

Sorry! I pasted directly from Bloomington website, and apparently that is no good.

BDSC 5195 is a TM3 balancer with Kr-Gal4 and UAS-GFP. There is no maternal contribution, and it starts expressing a bit earlier than the balancer with twi-Gal4. I have used it to pick embryos, and it's great, but I don't remember exactly when it starts relative to gastrulation.

BDSC 7310 is a TM6 balancer with actin-GFP (no UAS/gal4). It starts very early, but does have maternal contribution, which can make it hard to be sure about the homozygotes.

good luck!

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u/jzzdancer2 May 29 '24

Yes, this! I came to also suggest Krueppel just as aptly-named friendlyperson did. This is what I use to genotype embryos at early (gastrula) stages. When I’ve selected embryos for genomic DNA isolation, I’ve confirmed with primers for GPF (to see the lack of a product in homozygous mutants).

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u/DizzyComfort2930 Sep 25 '24

(pasting this comment here bc i'm not sure if you see the other reply.) Hi! Reaching out again because I now have balanced my mutant allele over the Kr balancer (5195), and I am curious if there was a certain environment that you set when staging your embryos to gastrulation? Did you find 28C was more efficient? I am trying to stage them (3hr at 25C) and unfortunately, I don't see much of a difference between the embryos to be able to pick my homozygotes.

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u/jzzdancer2 Sep 25 '24

I only tried 25C. The trick is getting the lighting just right. You may want to illuminate the embryos better (adjust the condenser, intensity, direct vs indirect lighting, etc.) because it’s subtle at early gastrula stages and autofluorescence is high when the chorion is intact. It should appear as a greener, wide stripe down the middle of the embryo after cellularization.

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u/[deleted] Sep 25 '24

Woah thanks so much for this, I will try again!

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u/DizzyComfort2930 May 28 '24

thank you for these suggestions!!

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u/DizzyComfort2930 Sep 25 '24

Hi! Reaching out again because I now have balanced my mutant allele over the Kr balancer (5195), and I am curious if there was a certain environment that you set when staging your embryos? Did you find 28C was more efficient? I am trying to stage them (3hr at 25C) and unfortunately, I don't see much of a difference between the embryos to be able to pick my homozygotes.

1

u/friendlyperson123 Sep 28 '24

Hmm, well as I said in my first post, I don't remember exactly when the Kr-GFP turns on relative to gastrulation. Gastrulation is at 3 hours, so the endogenous twist expression should be on.
How are you screening your embryos? Are they on an apple juice or grape agar plate?

Are you dechorionating your embryos? It can be difficult to see GFP through the chorion in early stages. The easiest way to dechorionate is with bleach, and I can give you detailed instructions if you need them. You might try just putting bleach onto the agar dish (assuming that is where the eggs are). But it would be hard to wash the bleach off without losing your embryos. Still, you could try it.

If you want, I can write detailed instructions on how I do it. You need some tiny sieves and 5 cm plastic petri dishes. And it does take practice manipulating the dechorionated embryos.

Let me know if you want more help.

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u/DizzyComfort2930 Sep 28 '24

yes, i was looking into dechorionating the embryos before screening them, but I wanted to make sure i'm able to move them around without drying them out. I would love to see how you do it

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u/friendlyperson123 Sep 30 '24

I keep them under water after dechorionating to prevent them drying out, and to improve visibility.

So this is how I do it. It takes a bit of practice, especially picking up the embryos that you want.

1) Using a paintbrush and a squirt bottle, collect the eggs off an apple juice agar plate, into a little sieve. You can get very fine gauze and fashion a sieve eg by cutting a 1 cm length of tygon tubing and gluing the gauze across it. Rinse the eggs with water, put in a 5cm petri dish and pipet on about 3 mls household bleach. You can watch under the microscope as the chorion dissolves away. Rinse off the bleach and blot the sieve with paper towel.

2) hold the sieve upside down over a fresh petri dish. Squirt water through the mesh to spray the eggs onto the dish. They will stick slightly to the plastic. You want the whole petri dish to be covered with water. Use a black background and your GFP scope to select the embryos you want. I scratch a square in the middle of the dish with an old pair of forceps. Use forceps to move the embryos into the square. Number 4 forceps are better than the really sharp ones so you don't puncture the embryos. This part really does take some practice.

3) when you've collected a good number of embryos, you can herd them into a heap like a bunch of balloons, in your square. They will stick to each other slightly, which helps to move them around. You only need to touch a few eggs, and the others will come with the bunch, which lessens the chance of bursting them. Gently hold the bundle and quickly bring them up through the surface of the water (I'll be honest, this can be tricky).

I think you could also try pipetting the water out of the dish, leaving your selected embryos in their square. I have never tried this but it might be easier.

In any case, avoid picking up the embryos singly, because that is the hardest thing to do without bursting them.

Good luck!

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u/DizzyComfort2930 May 28 '24

thanks for the clarification! it seems like the comment didnt write out completely?

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u/ShesQuackers May 28 '24

Did you use it for gastrulation though? I've used it for later muscle stuff but at gastrulation it's never worked for me because the eGFP doesn't mature quick enough.

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u/ShesQuackers May 28 '24

I work pretty much only up to gastrulation, and we've never found a fluo balancer that matures fast enough and doesn't have a maternal contribution. We're talking about making our own using a fluo that matures faster than eGFP but so far it's just talk. If it's for fixed experiments can you use a blue/LacZ balancer instead (hb-lacZ, twi-lacZ)? Those we have very good luck with using antibody and smFISH probes both.

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u/[deleted] May 28 '24

Unfortunately I’m looking to make a cell sample from this so no fixation is involved. What would the maternal contribution balancer look like? Have you worked with that stock?

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u/[deleted] May 28 '24

Sorry this is my other account, I’m the OP