Morpholino Oligo Injections
Morpholino oligonucleotides (MOs) (GeneTools) are a powerful approach to loss of function analysis in Xenopus embryos. Unlike dominant negative constructs, they have the advantage of increased specificity for the particular targeted gene.
In addition, X. tropicalis may have significant advantages over X laevis for the use of MOs. The allotetraploid genome of X. laevis may mean that the targeted gene may be represented by two slightly different copies commonly called an "A" form and a "B" form. These two forms of the same gene are likely functionally redundant but may vary enough in sequence to prevent the MO from binding effectively. Even one or two nucleotide differences can significantly reduce the effectiveness of the MO. X. laevis may then require two MOs to effectively knockdown a gene's function. X. tropicalis is diploid which should eliminate this problem. This complicated our analysis of beta-catenin (Khokha et al. 2002).
Additionally, as inbred strains are developed and more widely available, polymorphisms in the 5'UTR or near splicing sites should also be reduced. The shorter generation time of X. tropicalis makes this feasible.
Finally since genome sequencing of X. tropicalis is proceeding at a furious pace, it will become straightforward to design MOs against 5'UTR's or splice sites.
In our experience, we have observed phenotypes in X. tropicalis with a single MO when two MOs were required in X. laevis. This has faciliated combination knockdowns of two or more genes, another advantage of X. tropicalis for loss of function analysis with MOs.
Injection of MOs
We use our standard microinjection protocol for injecting MOs.
However there are some important things to consider in preparing your MOs especially if you want to avoid clogging your microinjection needle. First when obtaining MOs, we recommend resuspending in 0.1xMR to a concentration of 2-4 mM (add 75-150 microliters to 300 nM MO) or approximately 17-34 ng/nl. This concentration is an important consideration. In order to co-inject multiple MOs together, you have to be able to inject enough of each MO to give an adequate knockdown yet in a small enough volume not to be toxic to smaller X. tropicalis embryos. At the same time, MOs can precipitate causing the microinjection needles to clog.
It is worth noting however that MOs are not charged and cannot be precipitated with EtOH. If you do want to concentrate your MO, there are two options: either freeze-dry the MO or dry it in a Speed-Vac. You can then re-suspend the MO in a smaller volume and check the concentration using the following protocol. Since this is a bit complicated, choosing a starting concentration is an important consideration at the outset.
Once the concentration is determined, heat the solution to 65ºC for five minutes to ensure that the MO has gone into solution. Mix regularly. We typically make 5 aliquots and store at -20ºC. A working stock can be stored at 4ºC.
Prior to microinjection, warm the entire tube to 65oC for five minutes and then spin on a table top centrifuge at top speed for 2 min. Then aliquot the amount of MO that you intend to inject. In our experience, many morpholinos have a relatively low solubility and precipitate causing the microinjection needle to clog. Keep the MOs at 37ºC until your are ready to load your microinjection needle (we keep a warm block right next to our injection station). Recently, we have been heating them even further to 42oC without any harm to embryos and continued improvement in clogging. We find that MOs conjugated with fluorescein (which are convenient to trace) clog microinjection needles even more readily. By keeping the MO warm, we have greatly reduced our problems with needles clogging.
In general, we have often used doses of 20ng/embryo without any non-specific effects. In order to test this, we rescue with the appropriate mRNA co-injection. Therefore, it is helpful to keep the MO stock solutions RNase-free. MOs are extremely resistant to nucleases. However, dep treatment can harm MOs by reacting to the adenines of the MO. Therefore we have a 0.1xMR solution that is dep treated that we let stand for some time so the dep is completely gone. We resuspend our MOs in this and have found no problems. (Note: MOs are resistant to high temperature but are sensitive to acidic conditions.)
For an example, please see our paper on Spemann's Organizer.
Many thanks to Shannon Knuth of GeneTools for all her help with MOs
This page contributed by Mustafa K. Khokha