3.2.2 Germline Injection
1. Load 4 μL of DNA solution to the Femtotip II needle and
mount the needle to the microinjection station.
2. Load the embryo slide on the microinjection station.
3. Adjust the position of the needle to make sure that the embryos
and the tip of the needle are on the same focal plane.
4. Gently push the needle into the germplasm. The embryo does
not leak if appropriate drying time is applied.
5. Adjust the compensation pressure on the FemtoJet to avoid
absorption of germplasm into the needle. For a new needle,
50 hPa is desirable. Compensation pressure needs to be
adjusted during injection when the needle is worn or partially
clogged.
6. Adjust the injection pressure to five times as high as the compensation pressure and inject.
7. After all the embryos on the cover glass are injected, move the
cover glass into a humidity chamber saturated with water
vapor. Larvae are expected to hatch after 48 h at 18
C or
after 24 h at 25
C.
8. Pick the larvae using a dissecting needle and transfer them into
regular Drosophila food vials (20–30 larvae per vial). Avoid
halocarbon during the transfer.
9. When the adults hatch, cross them with appropriate balancer
lines (see Note 12). In the next generation, screen for the gain
or loss of selection markers (Fig. 2b, also see Note 13). PCR
genotyping is optional to confirm the edited region.
10. Two edited loci can be recombined in cis following standard
Drosophila genetics protocols. Screening for recombinants
should be performed 3 days after hatching if eye markers
are used.
3.3 Embryo
Preparation for Live
Imaging
1. Set up crosses to get triple-heterozygous females that supply all
three fluorescent proteins (MCP-3xtagBFP2, PCP-3xmKate2,
and ParB-eGFP).
2. Collect ~100 three-color virgin females and put them in an
embryo-collecting cup with ~50 males that carry the edited loci
(Fig. 2a).
3. Set up a sample holder with a piece of air-permeable membrane
as described in [9]. Add 50 μL of heptane glue on at the center
of the membrane. The glue should be completely dried when
the embryos are mounted.
4. Collect embryos at desired age on agar plates.
5. Cut a piece of double-sided tape (2 cm long) and push it
against the surface of the agar plates to adhere embryos on it.
378
Hongtao Chen and Thomas Gregor
1. Load 4 μL of DNA solution to the Femtotip II needle and
mount the needle to the microinjection station.
2. Load the embryo slide on the microinjection station.
3. Adjust the position of the needle to make sure that the embryos
and the tip of the needle are on the same focal plane.
4. Gently push the needle into the germplasm. The embryo does
not leak if appropriate drying time is applied.
5. Adjust the compensation pressure on the FemtoJet to avoid
absorption of germplasm into the needle. For a new needle,
50 hPa is desirable. Compensation pressure needs to be
adjusted during injection when the needle is worn or partially
clogged.
6. Adjust the injection pressure to five times as high as the compensation pressure and inject.
7. After all the embryos on the cover glass are injected, move the
cover glass into a humidity chamber saturated with water
vapor. Larvae are expected to hatch after 48 h at 18
C or
after 24 h at 25
C.
8. Pick the larvae using a dissecting needle and transfer them into
regular Drosophila food vials (20–30 larvae per vial). Avoid
halocarbon during the transfer.
9. When the adults hatch, cross them with appropriate balancer
lines (see Note 12). In the next generation, screen for the gain
or loss of selection markers (Fig. 2b, also see Note 13). PCR
genotyping is optional to confirm the edited region.
10. Two edited loci can be recombined in cis following standard
Drosophila genetics protocols. Screening for recombinants
should be performed 3 days after hatching if eye markers
are used.
3.3 Embryo
Preparation for Live
Imaging
1. Set up crosses to get triple-heterozygous females that supply all
three fluorescent proteins (MCP-3xtagBFP2, PCP-3xmKate2,
and ParB-eGFP).
2. Collect ~100 three-color virgin females and put them in an
embryo-collecting cup with ~50 males that carry the edited loci
(Fig. 2a).
3. Set up a sample holder with a piece of air-permeable membrane
as described in [9]. Add 50 μL of heptane glue on at the center
of the membrane. The glue should be completely dried when
the embryos are mounted.
4. Collect embryos at desired age on agar plates.
5. Cut a piece of double-sided tape (2 cm long) and push it
against the surface of the agar plates to adhere embryos on it.
378
Hongtao Chen and Thomas Gregor