Finally we provide molecular evidence for PB transposase act

Finally, we provide molecular evidence for PB transposase activity in vivo after plasmid intramuscular injection and electrotransfer of the murine muscles, for instance from the recovery and sequencing of several transposon–genomic junctions. Interestingly, such integration events were not able to mediate sustained transgene expression, as the GFP fluorescence declined for the muscles electroporated with the PB transposase expression vector. In contrast, GFP expression was more stable when the electrotransfer was performed in the absence of the transposase, which has been associated with the spontaneous genomic integration of episomal plasmid vectors in prior studies (Puttini et al., 2013). The cause of the lack of persistent expression from in vivo electroporated transposable vectors in the presence of the transposase remains unclear at present. It may include the persistence of low levels of the episomal transposase vector, leading to the remobilization of the transposons until they would either integrate into repressive chromatin structure or be lost from abortive transposition events. Alternatively, toxicity resulting from the persistent expression of the transposase episomal expression plasmid cannot be fully excluded, even if no signs of myofiber necrosis or regeneration were observed.
Acknowledgments This work was supported by grants of the Commission for Technology and Innovation of the Swiss Confederation and Selexis SA, the European Union Clinigene Network of Excellence in gene therapy and by the University of Lausanne. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Author contributions
Introduction Identification of adult intestinal stem cell markers has accelerated in recent years, following the discovery of the first bona fide marker, Lgr5, by Barker et al., 2007 (Barker et al., 2007). Powell et al. identified leucine-rich repeats and immunoglobulin-like domains protein 1 (Lrig1) as an intestinal stem cell marker in 2012 (Powell et al., 2012). At the same time, Wong et al. demonstrated that Lrig1 was important for intestinal homeostasis (Wong et al., 2012). While both groups demonstrated that Lrig1 marks DMOG in the intestinal epithelial stem cell zone, discrepant observations of Lrig1 protein distribution in the intestinal crypt were observed. Wong and colleagues, focusing on the small intestine, demonstrated that Lrig1 transcript and protein are expressed in the progenitor cell zone of the crypt base using in situ hybridization and immunofluorescent analysis. Using flow cytometry, they showed that 30% of intestinal epithelial cells express Lrig1 and these Lrig1+ cells express intestinal stem cell marker transcripts (Wong et al., 2012). Our group—focused on the colon—demonstrated that Lrig1 marks a bona fide intestinal stem cell population that gives rise to all differentiated intestinal epithelial cell types using lineage tracing studies. Additionally, we showed that Lrig1 protein is expressed in select cells in the colonic crypt base, rather than in a broad pattern. Flow cytometry demonstrated that only 4.8% of colonic epithelial cells express Lrig1; RNA-Seq analysis of this Lrig1+ flow-sorted population also revealed enrichment of intestinal stem cell marker transcripts (Powell et al., 2012). The relationship between different stem cell populations and between stem cells and committed progenitors, as well as studies of stem cell behavior, are marker-based. Therefore, it is essential to clarify the Lrig1 expression discrepancy to facilitate Lrig1-related studies. These two independent studies utilized different anti-Lrig1 antibodies to assess Lrig1 protein expression. Wong et al. used a commercial goat polyclonal anti-Lrig1 antibody from R&D Systems™, raised against nearly the entire ectodomain of mouse Lrig1 (#AF3688; hereafter anti-Lrig1-R&D) (Wong et al., 2012), while in collaboration with Covance (Denver, PA), Powell et al. generated a rabbit polyclonal peptide antibody to a sequence (KILSVDGSQLKSY) in the ectodomain of mouse Lrig1 (hereafter anti-Lrig1-VU) (Powell et al., 2012). Using a new Lrig1 reporter mouse (Lrig1-Apple), we set out to further characterize these antibodies to clarify their use for future Lrig1-related studies. We show that anti-Lrig1-R&D appears to recognize all Lrig1+ cells, while anti-Lrig1-VU recognizes a subset of Lrig1+ cells, likely expressing a non-glycosylated form of Lrig1.