The function of tcf3 in medaka embryos: efficient knockdown with pePNAs

Abstract

BackgroundThe application of antisense molecules, such as morpholino oligonucleotides, is an efficient method of gene inactivation in vivo. We recently introduced phosphonic ester modified peptide nucleic acids (PNA) for in vivo loss-of-function experiments in medaka embryos. Here we tested novel modifications of the PNA backbone to knockdown the medaka tcf3 gene.ResultsA single tcf3 gene exists in the medaka genome and its inactivation strongly affected eye development of the embryos, leading to size reduction and anophthalmia in severe cases. The function of Tcf3 strongly depends on co-repressor interactions. We found interactions with Groucho/Tle proteins to be most important for eye development. Using a dominant negative approach for combined inactivation of all groucho/tle genes also resulted in eye phenotypes, as did interference with three individual tle genes.ConclusionsOur results show that side chain modified PNAs come close to the knockdown efficiency of morpholino oligonucleotides in vivo. A single medaka tcf3 gene combines the function of the two zebrafish paralogs hdl and tcf3b. In combination with Groucho/Tle corepressor proteins Tcf3 acts in anterior development and is critical for eye formation.Electronic supplementary materialThe online version of this article (10.1186/s12896-017-0411-0) contains supplementary material, which is available to authorized users.

A simple and fast Agrobacterium-mediated transformation system for passion fruit KPF4 (Passiflora edulis f. edulis × Passiflora edulis f. flavicarpa)

“…Acetosyringone induces the activation and expression of virulence genes in Agrobacterium required for plant genetic transformation [ 43 ]. Depending on the plant species and Agrobacterium strain, the concentration of acetosyringone used for transformation varied from 20 to 200 µM [ 27 , 41 , 42 , 44 ]. In the present study, addition of acetosyringone into the Agrobacterium infection medium significantly increased the rate of transient gus A expression of inoculated leaf disc explants.…”

Environmental Assessment of Enzyme Production and Purification

“…An average protein yield of 21.35 ± 2.55 mg cGAS g CDW −1 was purified. The obtained value was assumed for the following calculations, as it seems to be reasonable in comparison to published values [ 19 , 20 ]. The estimation of the maximum cell concentration should rather be regarded as too low, since higher cell concentrations are to be expected especially in controlled bioreactor systems [ 4 ].…”

Effective refolding of a cysteine rich glycoside hydrolase family 19 recombinant chitinase from Streptomyces griseus by reverse dilution and affinity chromatography

“…Hence, we recommend that for every refolding trial, this critical point can be determined to attain optimum yield and efficiency. Mild refolding conditions involving the use of detergents [ 12 ], reversed micelles, and an intermediate or low concentration of denaturant [ 60 ] as well as physical conditions such as alkaline pH and/or high hydrostatic pressure have been reported [ 16 , 21 , 22 , 61 ] as strategies for high yields. HHP refolding has particularly been used successfully to refold several proteins including an oligomeric [ 17 ] as well as a disulphide rich protein [ 15 ] both of which demonstrated reasonably high yields of well folded protein.…”

“…HHP in the range of 1–3 kbar for about 90 mins, in combination with mild concentrations of chaotropes or alkaline pH have been reported to enhance dissociation of oligomers and disaggregation of intermolecular hydrophobic and electrostatic interactions [ 15 , 21 ]. Refolding is then promoted at low pressure of about 0.4 bar for up to 15 hrs [ 20 , 22 ]. While several successes have been reported with the use of HHP, the availability of equipment might be a major limitation to its immediate application for any refolding process.…”