Crossing Schemes
The generation time of Drosophila is about 10 days at 25o C. This is a crossing scheme for 10 weeks. The first generation is marked as F0, every following generation the number goes up. Section 6 will go through the individual generations step by step. This will serve as a reference and overview of the fly work to be done.
It is important to maintain a sufficiently sized (lots and lots of bottles) stock of the double balancer w, L/CyO; ftz, e/TM6B, Hu,Tb. The crossing scheme calls for female virgins of that stock at multiple times, and each and every cross should be done with 5-10 females. In F1, the yield of novel insertions is ultimately dependent on the number of offspring, and too few females in F2 won’t generate enough females of the right genotype for F3.
Scale of Crosses
As a general rule, 1-2 female could give rise to a productive cross. Double balancer females have a reduced egg production rate, so you need to take more of them. The more females, the more offspring, but you will need to watch overcrowding of vials. Flipping a cross every 2-3 days takes care of that. However, as a note of caution, flipping a puny cross is counterproductive.
Each team should consider the following targets:
F0: 5-10 individual crosses (separate vials). Each cross needs to be flipped 2-3 times. 4-8 virgin females, 2-3 males each
Harvest 40-80 F1 jumpstarter males
F1: 20-50 individual crosses (nobody stops you from doing more!). Each cross needs to be flipped 2-3 times. 8 virgin females, 2 jumpstarter males per cross
Harvest 10-40 (aka as many as possible) F2 single males with transposition event
F2: 10-40 single male crosses. Each vial needs to be flipped at least once. 5 female virgins, single male per cross.
F3: 3-5 virgin females per individual stock, 2-4 males of the same genotype. This is the most labor intensive step in terms of cross work!
F4: Flip individual stock vial.
Why do so many F1 crosses or even more? The tansposition frequency of P{w+,StanEx1} is about 1 in 100 – 1 in 150. From these, you can take males only that, in addition, have segregated the transposase away. So, compared to the number of flies you started with, you will end up with a small number of males in F2.
On the lower end of the scale, each team should get at least two good transposition events (red-eyed males that are non-Sb). We guestimate (transposition rate 1:100 – 1:150 for StanEx1) that approx. 600 flies need to be generated to get there. This translates back to at least 3 F1 crosses with 8-10 females each, and each individual cross at least flipped 3 times (every 2-3 days). Again, as from the experimentalist’s perspective the process of transposition is stochastic, there is no guarantee to actually see 2 good transposition males if the number of crosses is that low. The more individual F1 crosses are set up, the higher the chances of a good outcome.