Myelin-specific expression of the transgenes can be achieved with the rtn4a-

In the next step of the establishment of the transgenic lines, the Tg(hsp70:loxP-dsRed-loxP-egfp-T2A-rtn4a) lines were crossed to the mbp-specific driver line Tg(mbpa:mCherry-T2A-CreER^T2) to elicit restricted recombination in oligodendrocytes. In case of the rtn4a-loxP line, recombination could be achieved in all lines (6, 7 and 16). Recombination was effective and reliable foremost in line 6 (Fig. 7.6 D). To test whether recombination had adverse effects on the oligodendrocytes, double transgenic larvae were treated with 4-OHT and HS at 5 and 6 days, respectively, and analysed for fluorescence at 7 dpf. EGFP-positive larvae were selected and raised to an age of 18 dpf. At this stage, the heat shock was repeated without prior 4-OHT treatment. The fact that a large number of EGFP positive oligodendrocytes could be found in the spinal cord 24 h after heat shock showed, that the cells were not severely affected by the recombination.

When testing recombination in Tg(hsp70:loxP-dsRed-loxP-egfp-T2A-rat Nogo-A);

(mbpa:mCherry-T2A-CreER^T2) double transgenic larvae, however, no recombination could be observed. This was true for both available strains. Experiments were repeated several times and all different strains of the effector line were crossed with all available strains of the driver, but without success. Two possible explanations for the lack of recombination are conceivable to us. Either no recombination occurred or the expression of Nogo-A after the recombination proved to be fatal to the oligodendrocytes.

To analyse the events upon recombination, we examined larvae in short temporal intervals.

Every hour after heat shock on 4-OHT treated Tg(hsp70:loxP-dsRed-loxP-egfp-T2A-rat Nogo-A); (mbpa:mCherry-T2A-CreER^T2) double transgenic larvae, 20 larvae were selected and analysed under the confocal microscope.

130 Figure 7.6: Establishing the Nogo-A and rtn4a effector lines.

(A) Double transgenic Tg(hsp70:loxP-dsRed-loxP-egfp-T2A-rat Nogo-A); (ubi:CreER^T2) embryos only express Nogo-A after Cre activation by TAM and heat shock. Scale bar 500 µm. Nogo-A expression could be seen both in Tg(hsp70:loxP-dsRed-loxP-egfp-T2A-rat Nogo-A) transgenic embryos after heat shock that were injected with CremRNA in the one-cell stage (B) and in Tg(hsp70:loxP-dsRed-loxP-egfp-T2A-rat Nogo-A); (ubi:CreER^T2) double transgenic embryos that were treated with both TAM and HS (C). Nogo-A expression was only seen if both treatments were carried out. As a positive control, mouse brain lysate (M) was used. (D) Lateral view of a 7 dpf Tg((hsp70:loxP-dsRed-loxP-egfp-T2A-rtn4a);(mbpa:mCherry-T2A-CreER^T2) larva treated with 4-OHT and HS shows specific recombination in the oligodendrocytes of the brain and spinal cord.

Scale bar 500 µm.

This was performed from 1 h until 10 h post heat shock. Since the EGFP protein is formed faster than Nogo-A, green fluorescence should be visible in the cells at some point, regardless of whether or not Nogo-A is harmful. Nogo-A double transgenics showed no

131 fluorescence at any time during the observation, although Tg(hsp70l:loxP-DsRed-loxP-EGFP); (mbpa:mCherry-T2A-CreER^T2) double transgenic control larvae showed strong EGFP fluorescence in their oligodendrocytes within 5 hours after heat shock. One day after heat shock, there was still no EGFP fluorescence whereas the DsRed signal had fully developed by this time.

To receive further information about possible events in the genomes of the effector fish, the genome of the effector lines was directly analysed by PCR. A forward primer located in the hsp70 promoter and a reverse primer that binds in the EGFP gene were used. These primers would produce a 1500 bp band if the DNA stretch that is amplified was not recombined. After excision of the DsRed gene and the stop cassette, the PCR would produce a band of 300 bp in length. Recombination could therefore be visualised by a shift in the length of the PCR product from 1500 to 300 bp. The 300 bp band can be enhanced by choosing an elongation time that is insufficient to form the longer 1500 bp band (Fig. 7.7 A).

This could be applied in Tg(ubi:CreER^T2);(hsp70:loxP-dsRed-loxP-egfp-T2A-rtn4b) double transgenic embryos as well as Tg(mbpa:mCherry-T2A-CreER^T2 );(hsp70:loxP-dsRed-loxP-egfp-T2A-rtn4b) double transgenic larvae. As expected, 4-OHT/TAM treatment alone is sufficient to cause the shift in the length of the PCR product, since this is the treatment which leads to recombination (Fig. 7.7 B). Heat shock treatment alone does not lead to recombination.

While the PCR could show that the genome of the transgenic lines carrying either of the two zebrafish paralogues (dsRed-loxP-egfp-T2A-rtn4b) and (hsp70:loxP-dsRed-loxP-egfp-T2A-rtn4a)) is recombined both when crossed with the Tg(ubi:CreER^T2) and the Tg(mbpa:mCherry-T2A-CreER^T2) line, the Nogo-A effector construct was only recombined when crossed with the ubiquitous driver. No recombination was observed with the mbpa specific driver line, in accordance with the previous results.

In a third approach we tried to determine whether the number of oligodendrocytes is reduced in the spinal cord after recombination treatments, which would indicate that recombination took place and led to death of the cells. To this end, Tg(hsp70:loxP-dsRed-loxP-egfp-T2A-rat Nogo-A); (mbpa:mCherry-T2A-CreER^T2) adult fish were crossed to Tg(mbpa:EGFP) fish and the offspring screened for triple transgenic fish. As control fish,

132 double transgenic fish missing either the driver or the effector transgenes were used. All groups were submitted to recombination treatments and at 7 dpf, their spinal cord was analysed under the confocal microscope. Photos were taken and the number of oligodendrocytes in the spinal cord counted. This revealed no difference between triple transgenic fish and fish in which no recombination could have occurred due to lack of the driver or the effector element. In triple transgenic fish, 19.54 oligodendrocytes were found on average in a 300 µm long area of the spinal cord, compared to 19.57 in controls.

Figure 7.7: Genomic analysis by PCR visualizes recombination

(A) Theory of the recombination test by PCR. Primers that bind in the hsp70 promoter and the EGFP gene were used. In the unrecombined state the PCR produces a band of 1524 bp (a) or no band (b), if the elongation time is reduced. After recombination, the DsRed gene is absent from the DNA and a band of 305 bp is formed (c). If recombination occurs only in specific tissues and the elongation time long enough, the 1524 bp band can also still be present (d). (B) Recombination can be seen in all effector lines when crossed with the Tg(ubi:CreER^T2) driver line and treated with TAM (T) or TAM and HS (HS+T) (upper panel). No recombination is seen when embryos are DMSO treated or treated only with HS. When crossed with the Tg(mbpa:mCherry-T2A-CreER^T2) line, only the rtn4b and rtn4a effector lines show recombination after respective treatments. No recombination, however, can be seen in the Nogo-A effector line (lower panel).

Taken together, these results strongly indicate that recombination does not occur in Tg(hsp70:loxP-dsRed-loxP-egfp-T2A-rat Nogo-A); (mbpa:mCherry-T2A-CreER^T2) fish.

133 However, we will attempt to achieve recombination also in adult double transgenic fish since there might be a difference between developing and mature oligodendrocytes.