3.1.2 Integration of MS2
Stem-Loops, PP7 StemLoops, or parS Tags
Through RMCE
1. Construct the RMCE plasmid that contains the designated
stem-loops or parS sequence flanked by two attB sites (see
Note 10). If a deletion is generated from the CRISPR step,
the deleted genomic region should also be included in the
RMCE plasmid.
2. Set up embryo collection cages with germline-supplied phiC31
females and males that carry the Cas9-mediated attP knock-in.
Embryos collected from these cages are used for fly transformation. Follow Subheading 3.2 to generate transgenic flies
with the integrated imaging tags. Negative selection, i.e., loss
of the marker introduced with the Cas9-mediated knock-in, is
used to identify transformants.
3.2 Generation of
Transgenic Fly Lines
Transgenic fly lines are generated through germline injections.
In-home injections are recommended to facilitate fly maintenance
and speed up experiments. Different loci are generated separately
and genetically combined in cis or trans to create embryos that
carry multiple-edited loci.
3.2.1 Embryo Collection
and Mounting
1. Collect ~300 virgin females from the maternal Cas9 or integrase lines and put them together in an embryo collection
bottle with ~100 males that carry the targeted genomic loci.
Multiple collection bottles may be set up to increase embryo
yield.
2. 0–1-h-old embryos are collected on agar plates. 100–200
embryos are enough for a 1-h injection session.
3. To dissolve chorion, add 50% bleach onto the plates for 1 min.
Swirl gently every 20 s. Pour the de-chorionated embryos into
a plastic mesh well. Rinse with tap water for 1 min.
4. Transfer embryos onto a 60 Â 30 Â 10 mm 1% agar block and
align them perpendicular to the edge of the block. The anterior
tips of the embryos should face to the edge. Leave a space of
2 mm between the embryo tips and the edge of the agar block.
The adjacent embryos should be separated by ~1 mm. An
experienced injector is able to align 40 embryos in about 6 min.
5. Cover the long side of a piece of 50 mm cover glass with
double-sided tape. Adhere the aligned embryos to the tape
on the cover glass. The embryos’ posterior tips should point
to the edge of the coverslip after transfer.
6. Dry the embryos in the desiccant chamber. Drying time varies
between 0 and 5 min depending on seasons and weather conditions (see Note 11).
7. Take out the coverslip from the desiccant chamber and cover
the embryos with Halocarbon 200.
8. Repeat steps 4–7 for 20 min or until all embryos are mounted.
Imaging of Chromatin Loci and Transcription
377
Stem-Loops, PP7 StemLoops, or parS Tags
Through RMCE
1. Construct the RMCE plasmid that contains the designated
stem-loops or parS sequence flanked by two attB sites (see
Note 10). If a deletion is generated from the CRISPR step,
the deleted genomic region should also be included in the
RMCE plasmid.
2. Set up embryo collection cages with germline-supplied phiC31
females and males that carry the Cas9-mediated attP knock-in.
Embryos collected from these cages are used for fly transformation. Follow Subheading 3.2 to generate transgenic flies
with the integrated imaging tags. Negative selection, i.e., loss
of the marker introduced with the Cas9-mediated knock-in, is
used to identify transformants.
3.2 Generation of
Transgenic Fly Lines
Transgenic fly lines are generated through germline injections.
In-home injections are recommended to facilitate fly maintenance
and speed up experiments. Different loci are generated separately
and genetically combined in cis or trans to create embryos that
carry multiple-edited loci.
3.2.1 Embryo Collection
and Mounting
1. Collect ~300 virgin females from the maternal Cas9 or integrase lines and put them together in an embryo collection
bottle with ~100 males that carry the targeted genomic loci.
Multiple collection bottles may be set up to increase embryo
yield.
2. 0–1-h-old embryos are collected on agar plates. 100–200
embryos are enough for a 1-h injection session.
3. To dissolve chorion, add 50% bleach onto the plates for 1 min.
Swirl gently every 20 s. Pour the de-chorionated embryos into
a plastic mesh well. Rinse with tap water for 1 min.
4. Transfer embryos onto a 60 Â 30 Â 10 mm 1% agar block and
align them perpendicular to the edge of the block. The anterior
tips of the embryos should face to the edge. Leave a space of
2 mm between the embryo tips and the edge of the agar block.
The adjacent embryos should be separated by ~1 mm. An
experienced injector is able to align 40 embryos in about 6 min.
5. Cover the long side of a piece of 50 mm cover glass with
double-sided tape. Adhere the aligned embryos to the tape
on the cover glass. The embryos’ posterior tips should point
to the edge of the coverslip after transfer.
6. Dry the embryos in the desiccant chamber. Drying time varies
between 0 and 5 min depending on seasons and weather conditions (see Note 11).
7. Take out the coverslip from the desiccant chamber and cover
the embryos with Halocarbon 200.
8. Repeat steps 4–7 for 20 min or until all embryos are mounted.
Imaging of Chromatin Loci and Transcription
377