3.5.1.1 Dissection
Fertile quail eggs were incubated for 30-32 hours at 37°C up to stage 8-9 (HH) (Hamburger and Hamilton, 1992) corresponding to 4-6 somites so that the neural tube has not yet closed nor have the neural crest cells migrated (Fig. 3.1). Work was then carried out inside a laminar flow hood with flamed- sterilised instruments. The top part of the shell was cut out with a pair of scissors and the albumin removed carefully with forceps. The yolk was put on a petri dish with the pale halo on the top. A baked ring of filter paper was put on the yolk membrane encircling the embryo. The yolk membrane was cut around the filter paper containing the embryo, which was removed from the yolk and rinsed in cell culture medium (aMEM).
Fig. 3.1 Quail embryo at stage 8-9 (HH)
Anterior is to the left, posterior is to the right. The two dotted red lines demarcate the mesencephalic region from which cranial neural crest cells were dissected.
The fine dissection was then carried out on the stage of a stereomicroscope. The embryo was first dissected out of the membrane with iris scissors and was placed in a petri dish filled with medium. The mesencephalic region was cut out of the embryo then both neural crests were dissected using tungsten needle and set in 35 mm petri dish with the appropriate medium. The explants were incubated overnight at 37°C in 5% CO2, to allow the cells to settle down and to attach to the dish.
3.5.1.2 Transfection
When the lipid-mediated inteyrin targeted system (hart et a!., 1998) was used the following reagents, scaled up as required, were mixed and left at room temperature for 30 min.
LIpofectIn 6 pg (O.OIpg/pl in Optlmem) Peptide 6 32 pg (0.1 mg/ml in Optimem)
DNA 8 pg (optimum: 0.08 pg/pl In Optimem)
This was made up to 750 pi per dish with the appropriate volume of Optimem. The cells were washed In sterile PBS and then covered by the DNA complex. The dishes were Incubated for 2 h at 37°C in 5% CO2, before the mixture was replaced by aMEM medium. The transfection rate was assessed 24 h later by counting the total number of cells per culture compared with the number of fluorescent cells.
3.5.1.3 In ovo electroporation
The DNA solution was prepared by resuspending the required amount of precipitated plasmid in 8 pi of a solution of PBS calcium free supplemented with 1 mM Mg""". One microiitre of 1% carboxymethylcellulose was added to thicken the solution and 1 pi of fast-green was added to enable the solution to be visualised during injection into the embryo.
Fertile quail eggs were prepared as described in section 3.5.1.1. The yolk was put on a petri dish with the pale halo on the top. Using a glass capillary needle mounted on a cell microinjector, the DNA solution was injected through the yolk membrane In between the neural fold so that enough solution would fill up the forming neural tube. The tungsten electrodes were then immediately placed on the yolk membrane on each side of the embryo and 3 electric pulses of 50 Volts and 50 ms each were applied. The electrodes were inverted and the pulses applied in the other direction without moving the electrodes so that both neural crests were electroporated.
Immediately after electroporation, a baked ring of filter paper was laid out around the embryo and the niicrodissectiort was carried out as described above.
For wholemount embryo staining, the yolk containing the electroporated embryo was incubated for a further 24 h at 37°C so that development would carry on. Since quail shells are more brittle than the chick’s and can not therefore be windowed with Sellotape to prevent the yolk from drying out during incubation, the yolk was placed in a 35 mm dish and covered with the albumin. It was then placed in a bigger petri dish filled with water. After incubation, a large baked ring of filter paper was laid out around the embryo, which was then lifted and rinsed in PBS. The embryo was cut out of the yolk membrane and fixed overnight in 4% RFA for wholemount staining.
3.5.2 Cell lines
3.5.2.1 Defrosting
The cryogenic tube containing the cells was defrosted in a 37°C waterbath until the medium started melting. Five millilitres of warm medium was quickly added to the tube and the contents were transferred to a 15 ml falcon tube. The cells were centrifuged at 1000 rpm for 5 min in order to remove the DMSO harmful to the cells. The pellet was then resuspended in 5 ml of warm medium and put in a 25 cm^ flask to be incubated at 37°C in 5% CO2.
3.5.2.2 Passaging
When cells had reached confluence, they were washed twice in warm PBS and warm trypsin/EDTA was added. After 3 min, the flask was shaken briskly to detach the cells from the dish. Five millilters of warm serum-containing medium was added to inhibit the trypsin. The mixture was then transferred to a 15 ml falcon tube. After centrifugation for 5 min at 1000 rpm, the pellet was resuspended in an appropriate volume of warm medium to obtain a 6 fold dilution. The cell suspension was distributed evenly in 6 flasks that were topped up with medium to cover the cells.
3.5.2.3 Freezing
Sirniiariy to passaging, the cells were washed trypsined, centrifuged, and the pellet was resuspended in 2 ml of serum-containing medium. The number of cells was counted in an haematocytometer. After addition of DMSO to a final concentration of 10%, 10® cells per ml were distributed in cryogenic vials, which were left at -70°C overnight then stored in liquid nitrogen until used.
3.5.2.4 Electroporation
Cells were grown to 70% confluence, trypsinised as above, washed twice in PBS and counted. Approximately, 10^ cells were resuspended in 800 pi of PBS and placed in an electroporation cuvette together with 5 to 15 pg of DNA to transfect. The cuvette was left on ice for 10 min then electroporated at a specific voltage from 250 to 400 V and capacitance from 300 to 960 pFaraday. The content of the cuvette was split into 2 petri dishes filled with warm medium and incubated at 37°C in 5% CO2. After 48 h, selection was applied using the antibiotic Zeocin® (Invitrogen) at 200 ng/ml and the medium was changed every 3 or 4 days.