transport [6–8]. Two orthogonal systems (MS2 and PP7) have
been adopted for Drosophila [9–11]. Nascent mRNAs transcribed
from the tagged loci carry stem-loops at their 5
0 ends, preferably
intronic regions. These stem-loops are recognized by
corresponding genetically encoded coupling or coat proteins
fused to various fluorescent proteins, enabling visualization of the
physical locations and transcriptional activity of the tagged loci.
Furthermore, we introduced a DNA labeling approach based on
the bacteria DNA partitioning system (parABS). Here, a parS
sequence from Burkholderia was incorporated into the designated
loci [12–14] and maternally supplied ParB proteins then bound to
the parS sequence and aggregated to form diffraction-limited
spots.
In this protocol, we provide details regarding the construction
of CRISPR-edited flies, embryo preparation and mounting, choice
of fluorescent protein combinations for multicolor live imaging,
and optimized conditions of point-scanning confocal microscopy
for acquisition of high-quality 3D time-lapse images for localization
analysis. In particular, we discuss the relevant controls required to
assess the experimental errors associated with the measured distance between the tagged loci.
2 Materials
2.1 Fly Lines
1. Maternal Cas9 lines [15, 16] (see Note 1).
2. Maternal phiC31 integrase lines [17] (see Note 2).
3. Maternal lines that contain three fluorescent protein fusions for
three-color live imaging [18] (see Note 3).
2.2 Labeling
Endogenous Loci
1. Plasmids for CRISPR-based Drosophila genome editing [15]
(see Note 4).
2. Plasmids for recombination-mediated cassette exchange
(RMCE) [19, 20].
3. Plasmids for stem-loop series [7] (see Note 5).
4. Plasmids for parS-based genome labeling [12, 18] (see Note 6).
2.3 Genome Editing
and Recombination
1. Embryo collection cages.
2. Agar plates: 1% Agar in 20% Welch’s concord grape juice.
3. Mesh wells for embryo collection.
4. Halocarbon 200.
5. 50% Commercial bleach: 4.1% Sodium hypochlorite solution.
6. Double-sided tapes (Scotch, permanent).
7. Cover glass (22 Â 50 mm).
374
Hongtao Chen and Thomas Gregor
been adopted for Drosophila [9–11]. Nascent mRNAs transcribed
from the tagged loci carry stem-loops at their 5
0 ends, preferably
intronic regions. These stem-loops are recognized by
corresponding genetically encoded coupling or coat proteins
fused to various fluorescent proteins, enabling visualization of the
physical locations and transcriptional activity of the tagged loci.
Furthermore, we introduced a DNA labeling approach based on
the bacteria DNA partitioning system (parABS). Here, a parS
sequence from Burkholderia was incorporated into the designated
loci [12–14] and maternally supplied ParB proteins then bound to
the parS sequence and aggregated to form diffraction-limited
spots.
In this protocol, we provide details regarding the construction
of CRISPR-edited flies, embryo preparation and mounting, choice
of fluorescent protein combinations for multicolor live imaging,
and optimized conditions of point-scanning confocal microscopy
for acquisition of high-quality 3D time-lapse images for localization
analysis. In particular, we discuss the relevant controls required to
assess the experimental errors associated with the measured distance between the tagged loci.
2 Materials
2.1 Fly Lines
1. Maternal Cas9 lines [15, 16] (see Note 1).
2. Maternal phiC31 integrase lines [17] (see Note 2).
3. Maternal lines that contain three fluorescent protein fusions for
three-color live imaging [18] (see Note 3).
2.2 Labeling
Endogenous Loci
1. Plasmids for CRISPR-based Drosophila genome editing [15]
(see Note 4).
2. Plasmids for recombination-mediated cassette exchange
(RMCE) [19, 20].
3. Plasmids for stem-loop series [7] (see Note 5).
4. Plasmids for parS-based genome labeling [12, 18] (see Note 6).
2.3 Genome Editing
and Recombination
1. Embryo collection cages.
2. Agar plates: 1% Agar in 20% Welch’s concord grape juice.
3. Mesh wells for embryo collection.
4. Halocarbon 200.
5. 50% Commercial bleach: 4.1% Sodium hypochlorite solution.
6. Double-sided tapes (Scotch, permanent).
7. Cover glass (22 Â 50 mm).
374
Hongtao Chen and Thomas Gregor