Abstract
In this perspective article, we briefly review tools for stable gain-of-function expression to explore key fate determinants in embryonic brain development. As the piggyBac transposon system has the highest insert size, a seamless integration of the transposed sequence into the host genome, and can be delivered by transfection avoiding viral vectors causing an immune response, we explored its use in the murine developing forebrain. The original piggyBac transposase PBase or the mouse codon-optimized version mPB and the construct to insert, contained in the piggyBac transposon, were introduced by in utero electroporation at embryonic day 13 into radial glia, the neural stem cells, in the developing dorsal telencephalon, and analyzed 3 or 5 days later. When using PBase, we observed an increase in basal progenitor cells, often accompanied by folding aberrations. These effects were considerably ameliorated when using the piggyBac plasmid together with mPB. While size and strength of the electroporated region was not correlated to the aberrations, integration was essential and the positive correlation to the insert size implicates the frequency of transposition as a possible mechanism. We discuss this in light of the increase in transposing endogenous viral vectors during mammalian phylogeny and their role in neurogenesis and radial glial cells. Most importantly, we aim to alert the users of this system to the phenotypes caused by non-codon optimized PBase application in vivo.
Highlights
Much has been learnt about fate determinants in development during neurogenesis and gliogenesis from candidate approaches, natural gene mutations and genome-wide expression analyses
After reviewing different methods for stable gain-of-function expression systems, we show here an unexpected artifact of the piggyBac transposon system in the developing cortex
In this regard it is interesting to note that there seems to be a connection between the size of the transposon, ranging between 3.0 and 9.6 kBp, and the severity of the phenotype, as smaller transposons elicited a more severe phenotype than larger ones (Figure 2C and Supplementary Figure 1B)
Summary
Much has been learnt about fate determinants in development during neurogenesis and gliogenesis from candidate approaches, natural gene mutations and genome-wide expression analyses. The above results imply a striking difference between the effects of the codon optimized and the non-codon optimized version of the same enzyme Both proteins have the same amino acid sequence, but mPB has been found to enable an integration and transposition activity up to 20 times higher than PBase, likely due to a higher expression level in otherwise identical systems (Cadinanos and Bradley, 2007). To test if this is the case in the developing cortex, we measured the size and intensity of IUE at E18 for the mPB and PBase constructs combined with piggyBac-GFP. The promoter seems to have little effect on expression, and despite higher expression levels, mPB causes fewer developmental aberrations
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call