The mapping of SNPs made possible to design new morpholinos targeting se-quences without polymorphisms. For this purpose, I chose to use a splice-interfering MO against the intron1-exon2 splice site. In the case of the rx gene, the advantages of splice MOs over translation-blocking MOs are several. First, the efficiency of splice MOs is more easy to control, as this can be assayed via PCR. Unfortunately a good Rx antibody is not available yet, although this would be the perfect system to check the efficiency of MOs. Moreover, the rx 5’UTR is very polymorphic, making it almost impossible to identify a suitable MO target sequence.
A morpholino against the intron1-exon2 splice site, called rxin1ex2 MO, was in-jected at different concentrations in Platynereis zygotes. As a control, the stctrl MO provided by GeneTools (the company producing MOs) was injected at the same concentrations in sibling zygotes. To assess the effect of the rxin1ex2 MO on RNA splicing, total RNA was extracted from injected larvae and this was used to
pro-rx-ex2in2 MO
Figure 4.2: SNPs in the target sequences of Rx MOs. A. and B. show the distribution of SNPs in the target sequences of the first Rx MOs tested, rx-ex2in2 MO and rx-in2ex3 mo respectively. The alleles were identified after amplification and sequencing of several clones from different individuals (#1-7).
duce cDNA and then to amplify the rx gene (par. 12.3). From the rx gene structure and the position of the MO, the rxin1ex2 MO was expected to produce the retention of intron1 in the mature mRNA. Instead, I found that the rxin1ex2 MO produces the skipping of exon2 (fig. 4.3A-B). The consequence of this is the production of a shorter mRNA with a frameshift mutation and a premature stop codon; only a small part of the homeodomain is retained in this aberrant transcript.
The efficiency of knock down could be quantified with a qPCR assay (fig. 4.3C and par. 12.3). I used qPCR primers spanning the exon1-exon2 junction; these primers are designed to amplify specifically the normal transcript. Moreover, the total amount of rx transcript was measured with primer pairs in exon3 (present in both the normal and the aberrant mRNAs). In 48hpf injected larvae, there was up to a 75% downregulation of the normal rx mRNA. The efficiency of knock down was variable between injected batches, as a consequence of variability of injection volumes between different injection sessions.
In order to make different injection sessions more comparable, the injection proto-col was improved with the addition of TRITC-Dextran as injection tracer, and with the optimization of the injection needles, to deliver more consistent amounts of
in-wt no R
Figure 4.3: Aberrant splicing of rx mRNA after rxin1ex2 MO injection. A. Schematic drawing show-ing the position of rxin1ex2 MO, the aberrant splice product produced after rxin1ex2 MO injection, and the primer pairs used to analyze rx mRNA splicing. B. Gel picture of rx RT-PCR products, using a primer pair that amplifies the entire rx coding region. The rxin1ex2 MO injected larvae (48hpf) have two isoforms; the shorter one (arrow) lacks exon2 (407bp). C. Quantification of aberrant mRNA splic-ing with qPCR. In 48hpf injected larvae, the total amount of rx transcripts is not different between rxin1ex2 MO larvae and stctrl MO larvae (primer set 2, exon3). However, in the morphant larvae there is a significant reduction of the normal rx transcripts, recognized by a primer pair spanning the exon1-exon2 boundary.
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not mRNA
EGFP mRNA rx mRNA
EGFP mRNA rxin1ex2mostctrlmo
rxstcod2mostctrlmo
survival rate (%)
Figure 4.4: Survival rates of larvae injected with different morpholinos and mRNAs. The differ-ences between each experimental treatment and the controls are not statistically significant (t-test).
not mRNA injections (100-200 ng/µl): 6 experiments, 957 larvae, p=0.59; rx mRNA injections (312.5 ng/µl): 12 experiments, 2352 larvae, p=0.21; rxin1ex2 MO injections (800 µM): 43 experiments, 9144 larvae, p=0.21; rxstcod2 MO injections (500 µM): 11 experiments, 2155 larvae, p=0.89.
jection solution1.
In different series of experiments, I injected the mRNAs for not and rx and two different rx MOs (rxin1ex2 MO and rxstcod2, a MO targeting the 5’UTR). After each injection session, the embryos that failed the early cleavages were discarded. The embryos were also inspected for TRITC fluorescence, and the non injected embryos were eliminated. At the moment of fixation (usually 24 or 48hpf), I counted the num-ber of surviving larvae in the experimental and in the control groups (the controls were always injected either with stctrl MO or with the EGFP mRNA). The statistics are shown in fig. 4.4. For each treatment, the survival rate of control group was not significantly different from the survival rate of the experimental group. The sur-vival rate was correlated with the quality of the embryonic batch used for injection.
Injected larvae where the survival rate was lower than 50% were non considered for further analysis.
1Eppendorf Femtotips II were previously used, which break more easily; this caused higher variabil-ity in the injection volumes. Glass needles were tested and optimized with Dr. Mette Handberg-Thorsager. See also par. 12.2.2
mortality, compared to their controls. In all the experiments, the larvae of the control groups developed normally. Then, it can be assumed that the phenotypes observed in the experimental groups were specific and not caused by injection artifacts.
4.4. Establishment of rx knock out lines with the zinc finger nucleases