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    • We show that the PB transposon and its

    2018-11-12

    We show that the PB transposon and its transposase are functional and quite efficient in terms of stable gene transfer to primary mesoangioblasts when compared to the transfection of plasmid vectors. Transposition occurred even when using large transposons comprising two expression cassettes and a spacer or MAR element. The decrease of transposition efficiency observed with the larger transposons was paralleled by the lower transient transfection efficiency of the larger plasmids. This may therefore result from the known lower efficiency of the transfer of larger plasmids, which was previously shown to be inversely proportional to the size of the transfected DNA (Kreiss et al., 1999). This negative effect of large cargo DNA was reduced when using electroporation, a method that confers high transient expression but that may result in a lower frequency of integration of functional transgene copies. Here we show that the electroporation of the larger plasmids may lead to reduced expression despite the integration of many and probably often non-functional transgene copies in the absence of the transposase. However, expression of the transposase and vector transposition yielded low and homogenous copy numbers of well-expressed transgenes, and thus it nearly abolished these unwanted effects while allowing expression in 3–8% of the total cell population in the absence of any selection. Thus, transposition remained efficient in primary mesoangioblasts even when using transposons over 11kb, in agreement with studies performed on other types of undifferentiated primary K03861 Supplier (Ding et al., 2005). This study also shows that transposition can lead to higher expression levels per integrated transgene copy when compared to spontaneous plasmid integration, and that this high and persistent expression is mediated by few transposition events. Inclusion of a MAR element to reduce silencing effects was not accompanied by higher or more sustained expression in mesoangioblasts when placed next to potent promoter and enhancer sequences within the transposon vector, which contrasts with its requirement to achieve maximal transgene expression in cultured cell lines (Ley et al., 2013). This might reflect the less prominent silencing effects of undifferentiated primary cells, or a different tropism of the PB transposase in terms of the chromatin structure of the targeted genomic DNA in these cells. The PB transposase was proposed to preferentially integrate single copies of the transposon at genomic loci with a chromatin structure that is permissive for expression, whereas spontaneous plasmid integration occurs from rare recombination events of multiple copies of concatemerized plasmids with the cell genome, increasing the probability of transgene silencing (Folger et al., 1982; McBurney et al., 2002). The integration of a high number of plasmid copies was especially prominent following the antibiotic selection of cells having integrated plasmids in their genome by spontaneous recombination, as surviving clones expressing the antibiotic resistance gene often possessed a high plasmid copy number. This was not observed with the PB transposon vector, where the number of transgene copies remained within 1–3 copies, even following antibiotic selection, indicating a homogeneously low number of integrated transposon in the unselected polyclonal cell population. The propensity of some gene therapy viral vectors to integrate near the promoter or within the transcribed portion of cellular genes has been well documented (Gabriel et al., 2012; Nowrouzi et al., 2012). Whether the PB transposon may integrate preferentially at or near transcription units has also been the subject of several studies, and a preferential integration within or near transcribed genes has also been observed in some of the studies, although it may be influenced by the cell type and/or from biases introduced by the selection of cells expressing high levels of an antibiotic resistance gene (Wilson et al., 2007; Galvan et al., 2009; Huang et al., 2010). Here we found that the transposition of PB vectors may not occur preferentially in the genes of unselected mesoangioblast populations, since 73% of the recovered integration events were found in intergenic regions, mainly beyond 10kb from the nearest transcribed gene sequence, although genic integration events were also observed. Furthermore, we demonstrated the feasibility of obtaining mesoangioblast clonal populations expressing GFP at stable levels from a single intergenic transposon integration site. These stable mesoangioblast clones maintained their ability to differentiate in vivo and to mediate GFP expression. These findings support the rationale of using PB transposon vectors to develop stem cell based therapeutic approaches.