The methods described in this section were performed according to Sambrook et al.
(1989) if not otherwise stated.
4.3.1 Preparation of chemically competent E.coli cells
Competent E. coli cells of the Top10 strain (Invitrogen GmbH) were prepared according to the protocol by Inoue et al. (Inoue et al. 1990) and checked for competence by a test transformation with the pBluescript KS (-) plasmid. The cells were stored at -80°C until use.
4.3.2 Transformation of chemically competent E.coli
Transformation of E.coli was performed by adding 100 µl of competent cells to a maximum amount of 10 µl of DNA solution. The cells were incubated on ice for 30 min and heat shocked at 43°C for 1 min. Afterwards they were chilled on ice for 1 min and 1 ml LB medium was added. Subsequently they were incubated for 1 h on a 37°C shaker and plated on an agar plate containing the appropriate selection antibiotic. E.coli cells were grown over night at 37°C in an incubator.
4.3.3 Cryopreservation and recovery of E.coli clones
0.5 ml of an E.coli overnight culture were mixed with 0.5 ml of 100% glycerol in a reaction tube and submersed in liquid nitrogen. Subsequently the sample was stored at -80°C until reuse. In order to regrow the culture approximately 50 µl of the frozen stock were grown for 12 h in 5 ml LB medium for miniprep DNA purification or for 16 h in 200 ml LB medium for maxiprep purification.
4.3.4 Plasmid isolation
Bacterial cells were grown from single colonies or glycerol stocks. Small-scale plasmid isolation was performed using 5 ml of an E. coli overnight culture using the Qiagen Spin Miniprep kit according to the manufacturer’s instructions. Finally the DNA was eluted in 50 µl of buffer.
For large-scale plasmid preparations 250 ml of E. coli liquid culture were grown over night. Plasmid DNA was extracted with the Qiagen Plasmid Maxi kit according to the manufacturer’s protocol.
4.3.5 DNA restriction digest
For analytical DNA digestions 2 µl (app. 500 ng) of DNA were incubated with 2 units (one unit cleaves 1 µg of DNA per hour) of the appropriate restriction enzyme. The incubation was carried out in the reaction buffer and at the temperature recommended by the manufacturer for 1 h.
For preparative digestions 10 µg of DNA were incubated with 10 units of the restriction enzyme for at least three hours at the appropriate reaction temperature. If a double digestion was not possible according to the manufacturer’s recommendations, the DNA was ethanol precipitated and resuspended in nuclease free water prior to the second restriction digest.
4.3.6 Alkaline phosphatase treatment
To prevent religation of vector DNA leading to false positive colonies, the DNA was treated with shrimp alkaline phosphatase after restriction digestion. Therefore 0.5 units of the phosphatase enzyme were added to the restriction digest per 10 µg of DNA. Then the reaction mixture was incubated for 15 min at 37°C.
4.3.7 Agarose gel electrophoresis
0.5-2 % (w/v) of agarose were dissolved in 1x TAE buffer by heating in a microwave oven. After cooling the agarose solution for 5 min 2 µl of 1 % (w/v) ethidiumbromide solution were added per 100 ml of agarose. The solution was poured into a gel tray and a comb was inserted. After solidification the comb was removed and the gel was put into a chamber filled with running buffer. DNA running buffer was added to the samples, which were subsequently loaded onto the gel together with the DNA ladder.
Then an electrical current was applied. Segregation of DNA bands was checked under UV light. Gel pictures were taken with a Gel Jet Imager (Intas GmbH).
4.3.8 Purification of DNA from agarose gels
DNA fragments were extracted from agarose gels after the size could be clearly determined and they have reached a sufficient segregation. The DNA bands were cut out from the gel and the DNA was purified with the QIAquick gel extraction kit
Vector and insert DNA concentration were determined as described in section 4.3.9 Reaction mixtures were prepared with molar vector to insert concentrations of 1:3, 1:5 and 1:10. Subsequently T4 DNA ligase and reaction buffer were added. The reaction was incubated at room temperature for sticky end ligations or at 16°C for blunt end ligations for 2 h.
4.4 Immunohistochemistry
4.4.1 Cryosectioning of mouse spinal cords and embryos
Mice were sacrificed by decapitation or cervical dislocation. Subsequently the animals were eviscerated and the spinal cords were removed and collected in ice-cold PBS. Afterwards the cords were fixed by immersion in 4 % paraformaldehyde solution. The fixation time was 6 h for spinal cords from mice up to P10 and over night for spinal cords from older animals. Then the spinal cords were washed for 24 h in PBS followed by dehydration in PBS containing 30 % Sucrose for 6 h. All incubation steps were carried out at 4°C on a vertical shaker. After dehydration the spinal cords were equilibrated for 5 min in OCT and placed into an embedding mold filled with OCT. The embedding molds were placed on dry ice in order to solidify the OCT. Embedding molds were stored at -20°C or -80°C until cutting.
For cutting the solidified OCT blocks were removed from the molds and mounted in a CM 1510 S cryostat (Leica Microsysteme GmbH). The tissue was cut into 30 µm slices for postnatal mouse spinal cords and chicken embryos and 20 µm slices for mouse embryos. The slices were attached to SuperFrost Plus cover slides and air dried for 1 h at room temperature. Subsequently they were stored at -20°C until use.
4.4.2 Cryosectioning of chick embryo spinal cords
The eggs were chilled at room temperature for 1 h to reduce movement of the embryos. Subsequently the embryos were removed and decapitated. The vertebra was extracted and washed in cold PBS. Then the vertebra was fixed in 4 % paraformaldehyde for 6 h (E12) or 8 h (E18). The subsequent steps were the same as described in 4.4.1 for mouse spinal cords.
4.4.3 Immunohistological staining procedure
Cryosections were washed two times for 10 min in PBS in order to rehydrate and to remove residual embedding material. Afterwards the primary antibody solution is applied. Primary antibodies were diluted in PBS pH 7.2 containing 1 % BSA and 1 % Triton X-100 to the concentration given in Materials (3.2.1). The sections were incubated at 4°C over night.
The next day the primary antibody solution was removed and the sections were washed trice with PBS for 10min each. Afterwards the appropriate secondary antibodies were applied in the concentrations given in Materials (3.2.2) in antibody staining solution. The sections were incubated for 1 h at room temperature.
Afterwards they were washed trice with PBS and mounted with VectaShield or 50 % glycerol in PBS.