Open Access Research article

Side chain modified peptide nucleic acids (PNA) for knock-down of six3 in medaka embryos

Sebastian Dorn1, Narges Aghaallaei1, Gerlinde Jung2, Baubak Bajoghli13, Birgit Werner4, Holger Bock4, Thomas Lindhorst4 and Thomas Czerny12*

Author Affiliations

1 Department for Biomedical Sciences, University of Veterinary Medicine, Veterinärplatz 1, A-1210, Vienna, Austria

2 Department for Applied Life Sciences, University of Applied Sciences, FH Campus Wien, Helmut-Qualtinger-Gasse 2, A-1030, Vienna, Austria

3 Current address: Director’s Research Unit, European Molecular Biology Laboratory (EMBL), Meyerhofstrasse 1, 69117, Heidelberg, Germany

4 Ugichem GmbH, Mitterweg 24, A-6020, Innsbruck, Austria

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BMC Biotechnology 2012, 12:50  doi:10.1186/1472-6750-12-50

Published: 17 August 2012

Additional files

Additional file 1:

Figure S1. Building blocks used for the chemical synthesis.

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Additional file 2:

Table S1. Optimisation of the PNA length. The names of the PNAs are explained in Figure 2. The embryos were injected with a mixture of 10 ng/μl gfp mRNA and 200 μM PNAs preincubated for 30 minutes on ice. After 24 hours the embryos were divided into groups according to their gfp signal intensity. The average gfp intensity of the surviving embryos was then calculated as described in the text.

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Additional file 3:

Table S2. Antisense function of mixed PNAs on gfp mRNA. Injections and evaluations were performed as described in Table 1, except that 20 ng/μl mRNA was used. Note that the increased amount of mRNA results in higher numbers of average gfp intensity for comparable antisense function.

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Additional file 4:

Figure S2. Distribution of mix-PNAs in the embryo. Medaka embryos injected with 100 μM solution of Gfp16mixRho are shown in the 2-cell stage (A) and stage 24 (16 somites; B). A dashed line indicates the outline of the yolk. A lateral view of the embryo is shown in B.

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Additional file 5:

Table S3.Six3 knock down by PNAs. PNAs and morpholino oligos were injected at the indicated concentrations and the embryos evaluated after 3 days at stage 29 according to the severity of the phenotype. “Phenotypes in surviving” indicates the percentage of surviving embryos showing Six3 phenotypes.

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Additional file 6:

Figure S3. Comparison of mixed PNA and morpholino phenotypes. Embryos were injected with 400 μM Six3mix-PNA (PNA) or 100 μM Six3-MO (MO) and at stage 20 analysed by in situ hybridisation with probes for pax2 and rx2. Typical results are shown for each group of weak, moderate and strong phenotypes, wildtype (WT) embryos are shown as a reference. An arrowhead marks pax2 expression in the mid-hindbrain boundary, note the reduced expression in the strong group. The eye vesicles in the weak group are smaller compared to wildtype embryos, in the moderate group their size is further reduced, in the strong group no eye vesicles were detectable.

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Additional file 7:

Figure S4. Rescue of Six3mix-PNA injected embryos. Embryos were injected with 400 μM Six3mix-PNA (PNA) together with gfp-mRNA (10 ng/μl) or hSix3-mRNA (5 and 10 ng/μl). The phenotypes were then determined according to criteria described in the text. “Phenotypes in surviving” indicates the percentage of surviving embryos showing six3 phenotypes.

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