8. embed with Eukitt
nerve at the site where the forceps have been applied indicates a successful crush of the nerve.
A successful cut is indicated by a visible retraction of the nerve bundle separated from the eye. For the enucleation experiments, individuals are anaesthetized as described above and the left eye is removed using fine scissors. After surgery, gills are flushed with tank water by gently pulling the fish through the water. Fish resume breathing within a few seconds.
2.20.2.2 Spinal Cord Transection
Spinal cord transections are performed as described previously (Becker, Wullimann et al.
1997). Briefly, fish are anaesthetized, scales are removed from the flanks of the fish at the level of the spinal cord, a longitudinal incision is made and the vertebral column is exposed by pushing the muscle tissue aside. Then the vertebral column is cut with micro-scissors at a level halfway between the dorsal fin and the operculum, corresponding to the eighth vertebra.
The wound is sealed with a drop of histoacryl (B. Braun Melsungen, Germany). After surgery, fish are revived as described above for optic nerve lesions.
2.20.3 Perturbation of Gene Expression by Morpholinos
2.20.3.1 Injection of Morpholino into Freshly Fertilized Eggs
The zebrafish offers the possibility to analyze the functions of genes during early development in vivo by injecting reagents directly into the freshly fertilized egg. Antisense oligonucleotides are widely used to inhibit the translation of proteins in a variety of model systems. The first antisense oligonucleotides like RNA and single-stranded DNA were derived from natural origins. Later developed oligonucleotides were chemically synthesized and several modifications were added to improve stability and specificity (Summerton and Weller 1997). Morpholino phosphorodiamidates, a new generation of antisense oligonucleotides which were originally developed for therapeutic approaches (Arora, Knapp et al. 2000) are the first viable sequence specific gene inactivation method in the zebrafish.
Morpholinos are synthetic DNA analogues which contain a morpholine ring in lieu of the standard ribose sugar moiety and contain a neutral backbone. Morpholinos are very resistant to a variety of nucleases, they show a low toxicity and have a high affinity to RNA (Summerton et al., 1997). Morpholino phosphorodiamidates function in a RNAse H independent manner, which makes them different from other antisense oligonucleotides. A morpholino selected against the leader sequence or nearby bases of an mRNA can bind to it
and sterically inhibit scanning of the mRNA by the 40S ribosomal subunit (Ekker and Larson 2001). The efficacy of morpholinos is restricted to target sites within the leader and sequences surrounding the start codon (Summerton 1999), a bound morpholino does not appear capable of altering activity of the ribosomal complex once translation is initiated. Thus, only a small fraction of the transcribed RNA sequences bound by morpholinos within a cell will result in a deleterious effect on gene function.
A different possibility are splice site directed morpholinos. Morpholino oligonucleotides can block nuclear processing events, pre-mRNA processing in particular. The power of high specificity and steric blocking allows one to specifically and reproducibly delete exons of choice by blocking access of the splicing machinery to the pre-mRNA. This technology, not possible with RNase-dependent or RISCdependent oligonucleotides (phosphorothioates, RNAi and others), not only allows characterizing specific exon function and creating loss-of-function deletions or insertions but it also allows researchers to eliminate a specific splice variant while leaving another splice variant of the same gene intact. Many papers have now been published using morpholinos to block splicing by targeting exon-intron or intron-exon boundaries (Draper, Morcos et al. 2001).
Since protein synthesis of specific genes is not completely inhibited by morpholino application, the effect will be referred to as a knockdown.
To establish morpholino use in the zebrafish, phenotypes of known mutants such as no-tail (Schulte-Merker, van Eeden et al. 1994) were copied. The morpholinos were injected into fresh fertilized eggs and shown to phenocopy the mutations (Nasevicius and Ekker 2000).
Morpholinos have been used with great success in many studies to gain information of gene function (Erter, Wilm et al. 2001; Solomon and Fritz 2002). They are also useful to analyze gene function in the fruit fly Drosophila melanogaster, the frog Xenopus laevis and in cell culture systems (Heasman 2002). Unspecific effects of morpholinos were also described (Nasevicius and Ekker 2000), these effects are due to hybridization of the morpholino to sequences similar to the target or are dose dependent (Ekker and Larson 2001).
300 nmol lyophilized morpholinos specific for robo2 (see appendix for sequences) were delivered and resuspended in 41.5 µl Danieau solution to obtain a stock solution of 65ng/nl (8mM). The stock solutions were split in 3 µl aliquots and stored at –20°C. Morpholinos were adjusted with Danieau solution to obtain a concentration of 8ng/nl and were injected into 1-4 cell staged eggs as described below.
Morpholino injection:
Freshly fertilized eggs are harvested and disinfected with autoclaved fish water containing 0.02 % methylenblue (500Xl/1Liter).
Eggs are washed three times with the treated fish water and arranged in a groove in a petri dish containing 2 % agarose. Usually 0.5 µl 5% rhodamine dextran (MW = 10000) are added to a 3 µl aliquot of morpholino solution to visualize the amount of injected liquid. A glass micropipette is filled with the solution by capillary forces and held by a micromanipulator.
Morpholino is injected with a Microinjector (Narishige Intracel, Japan) directly into the yolk of 1-4 cell staged eggs. Finally, injected eggs are incubated in the autoclaved fish water plus methylenblue at 28.5°C until the desired developmental stage is reached and embryos are subjected to phenotypic analysis.
2.20.3.2 Using a Gelfoam Soaked with Morpholino
To influence gene translation in adults, especially in the regeneration of the optic nerve, a new method for specific morpholino delivery is used. To transport the morpholino specifically into the RGC layer the method by Goldmann (Veldman, Bemben et al. 2007) is used, who himself adapted this method from a spinal cord lesion paradigm by the Becker lab using a morpholino soaked gelfoam.
Immediately after a transection of the optic nerve (without damaging the blood vessels supplying the eye!) directly behind the eye, a piece of Gelfoam soaked with a defined amount of morpholino is placed on the nerve stump and the eye is carefully put back into its socket.
Detailed description of the operation to be found in section: 2.20.2.1 Protocol for the preparation of morpholino soaked Gelfoam pieces:
1. place a small Petri dish (35 x 10 mm) onto a piece of Parafilm and circle with a marker around the dish
2. cut a round piece of Parafilm and place it in the Petri dish 3. place a small chunk of Gelfoam into that Petri dish
4. use the watchmaker’s forceps to tear pieces (one for each operation; plus one extra as a backup) off the Gelfoam chunk which have approx. the diameter of the optic nerve – chunks will shrink a bit when solution is soaked up – and remove the remaining big chunk from the Petri dish
5. prepare the morpholino dilution (in my case: 8,16 and 32Xg/Xl) in Danieau solution 6. pipette 1Xl drops of diluted morpholino solution next to each Gelfoam piece in the
Petri dish onto the Parafilm
7. carefully pull each Gelfoam piece into its morpholino drop and let it soak up the liquid (Tip: use a pen to make a small dot next to the soaked up Gelfoam – they are hard to see once they are dried up)
8. after 1 or 2 minutes the soaked Gelfoam piece dries up and is ready to be placed on the nerve stump behind the eye (they are easier to handle if they are still a bit wet)
All soaked Gelfoams were used and none stored.