8. Drying chamber (15 Â 10 Â 5 cm) filled with Drierite desiccant (3 cm in depth).
9. Femtotip II microinjection needles (Eppendorf).
10. FemtoJet pump (Eppendorf).
11. Microinjection station.
12. Humidity chamber.
13. Drosophila food vials.
14. Fluorescence stereoscope for phenotyping.
2.4 Embryo Live
Imaging
1. Embryo collection cages.
2. Agar plates: 1% Agar in 20% Welch’s concord grape juice.
3. Ultrathin air-permeable membrane (Lumox film 25 um, Sarstedt Inc.).
4. 3D printed membrane holders [21].
5. Dissecting needles.
6. Heptane glue (see Note 7).
7. Halocarbon 27.
8. Cover glass (18 Â 18 mm, No. 1.5).
9. Confocal microscope with multiple laser lines and highsensitivity photon detectors.
10. Three-color-coated TetraSpec beads (200 nm).
11. Autofluorescent plastic slide (Chroma) for flat-field
adjustment.
3 Methods
We provide detailed protocols for generating fly lines that carry
multiple-edited genomic loci. Crossing males from these lines with
the three-color females will produce embryos for multicolor live
imaging. We advocate strict controls during sample preparations
and live imaging procedures in order to obtain high-quality and
reproducible image data for quantitative analysis.
3.1 Editing of
Endogenous Genes to
Contain MS2, PP7, and
parS Tags
Cas9-mediated homologous recombination is used to integrate
RNA stem-loop sequences or parS sites at the target gene loci. To
enhance the flexibility of the in vivo design, an intermediate step
using recombination-mediated cassette exchange (RMCE) is
adopted (Fig. 1a).
3.1.1 Cas9-Mediated
Knock-In
1. Select a guide RNA (gRNA) target site that contains a protospacer adjacent motif (PAM, 5
0 -NGG-3
0 ) [15]. The PAM site
should be as close to the targeted locus as possible. Two gRNA
Imaging of Chromatin Loci and Transcription
375
9. Femtotip II microinjection needles (Eppendorf).
10. FemtoJet pump (Eppendorf).
11. Microinjection station.
12. Humidity chamber.
13. Drosophila food vials.
14. Fluorescence stereoscope for phenotyping.
2.4 Embryo Live
Imaging
1. Embryo collection cages.
2. Agar plates: 1% Agar in 20% Welch’s concord grape juice.
3. Ultrathin air-permeable membrane (Lumox film 25 um, Sarstedt Inc.).
4. 3D printed membrane holders [21].
5. Dissecting needles.
6. Heptane glue (see Note 7).
7. Halocarbon 27.
8. Cover glass (18 Â 18 mm, No. 1.5).
9. Confocal microscope with multiple laser lines and highsensitivity photon detectors.
10. Three-color-coated TetraSpec beads (200 nm).
11. Autofluorescent plastic slide (Chroma) for flat-field
adjustment.
3 Methods
We provide detailed protocols for generating fly lines that carry
multiple-edited genomic loci. Crossing males from these lines with
the three-color females will produce embryos for multicolor live
imaging. We advocate strict controls during sample preparations
and live imaging procedures in order to obtain high-quality and
reproducible image data for quantitative analysis.
3.1 Editing of
Endogenous Genes to
Contain MS2, PP7, and
parS Tags
Cas9-mediated homologous recombination is used to integrate
RNA stem-loop sequences or parS sites at the target gene loci. To
enhance the flexibility of the in vivo design, an intermediate step
using recombination-mediated cassette exchange (RMCE) is
adopted (Fig. 1a).
3.1.1 Cas9-Mediated
Knock-In
1. Select a guide RNA (gRNA) target site that contains a protospacer adjacent motif (PAM, 5
0 -NGG-3
0 ) [15]. The PAM site
should be as close to the targeted locus as possible. Two gRNA
Imaging of Chromatin Loci and Transcription
375