$$\rightleftharpoonup{xx}$$
$$\longleftharp{xx}$$,
$$\longrightharp{xx}$$,
This protocol is an excerpt from Du et al., An Efficient Strategy for Generating Tissue-specific Binary Transcription Systems in Drosophila by Genome Editing, J. Vis. Exp. (2018).
1. Fly genetics and screening (Figure 1 and Figure 2)
- When the injected embryos develop into adults, cross each single G0 flies to balancer flies. Select suitable balancer for the chromosome containing the targeted allele.
- Anesthetize the F1 offspring from each G0 cross on a CO2 pad and randomly pick 10-20 males under a stereomicroscope. Cross them individually to the balancer females as shown in Figure 2.
- When the F2 larvae hatch, pick the single F1 father from each cross and extract gDNA using the single fly genomic DNA preparation protocol:
- Prepare gDNA extraction buffer: 10 mM Tris-Cl pH 8.2, 1 mM EDTA, 25 mM NaCl, store at room temperature. Prepare 20 mg/mL Proteinase K stock solution and store in the freezer.
- Put each fly in a 1.5 mL micro-centrifuge tube and label the tube. Keep in the -80 °C freezer overnight.
- Prepare a fresh working volume of gDNA extraction buffer containing 200 µg/mL final concentration of Proteinase K.
Note: Do not use an old buffer for this step.
- Squish each fly for 10–15 s with a pipette tip containing 50 μL of squishing buffer without dispensing the liquid. Dispense the remaining buffer into the tube and mix well. Incubate at 37 °C for 20–30 min.
- Put tubes in 95 °C heat block for 1–2 min to inactivate the Proteinase K.
- Spin down for 5 min at 10,000 x g. Store the preparation at 4 °C for further PCR analysis.
- Use the same method to prepare gDNA from a nos-Cas9 fly, which serves as a negative control. Perform three-step PCR based screens to identify the correct "ends out" HDR (Figure 1, Figure 3) using gDNA of each F1 male as a template5. Use 1 μL of the DNA prep in the following PCR reaction system: 10 μL of 2x PCR Master Mix with Dye, 1 μL of each primer (10 μM), 1 μL of DNA template, and 7 μL of ddH2O.
- As shown in Figure 3A, perform PCR using primers fwd1 and rev1 to screen for the existence of the insertion or replacement; perform PCR using fwd2 and rev2 primers to verify the insertion or replacement from 3' region; perform PCR using primers M13F and rev3 to check "ends-in" HDR (Table 1C).
- Keep the fly lines with the confirmed ends-out HDR and establish balanced stocks from the F2 generation. Outcross to the balancer flies again to remove any unintended mutations on other chromosomes.
