wnt signaling pathway Plasmid electroporation EP to the brai

Plasmid electroporation (EP) to the wnt signaling pathway has become a commonly used method of in vivo transgenesis in development (Breunig et al., 2007; Saito and Nakatsuji, 2001). However, like most episomal methods of transgenesis, EP suffers from plasmid dilution (Chen and LoTurco, 2012). Thus, studying complex lineage trees or postnatal neuro- and gliogenesis has been limited (Chen and LoTurco, 2012). To mitigate this, the recently characterized piggyBac transposon system permits stable integration of transgenes and can be used with various standard in vitro transfection methods to generate stable cell lines (Chen and LoTurco, 2012; García-Marqués and López-Mascaraque, 2013; Kahlig et al., 2010).
Results We constructed a custom genetic system for stable Tet-regulated flexible transgene expression using piggyBac transposition, termed pB-Tet-GOI (piggyBac-transposable Tet-mediated flexible expression of a genetic element of interest). pB-Tet-GOI consists of three plasmids: (1) a pBase-expressing plasmid for transposon integration, (2) a reverse Tet transactivator (rtTA) plasmid (or transactivator variants [tTA2 and tTA2-CA] discussed below) to allow for dox-regulation of the GOI, and (3) a Tet response GOI plasmid (Figure 1A). This tripartite system provides flexible, “mix and match” use of the different tTA2/rtTA variants with different GOI response plasmids. pBASE itself recognizes the piggyBac terminal repeats (TRs) that flank the rtTA and response plasmids and thereby catalyzes stable genomic insertion of these plasmids to avoid plasmid dilution with cell division (Chen and LoTurco, 2012). Importantly, the pBase plasmid is episomal and dilutes with cell division, preventing the stably inserted rtTA and response plasmids from continuously “hopping” in and out of the genome. The rtTA plasmid yields constitutive expression of rtTA-V10 protein. With dox, the rtTA-V10 binds to the Tet-Bi promoter of the response plasmid and induces bi-directional transcription of two genes of interest (i.e., “Tet-On”). Finally, the Tet response plasmid features a constitutively expressed TagBFP2 reporter with a nuclear localization signal (NLS) and a V5 epitope tag (Figure 1A) for fluorescent and immunocytochemical (ICC) identification of transgenic cells. In addition, this design was employed to minimize spurious Tet-responsive gene expression by using the TagBFP2 cistron as a genetic insulator, minimizing possible upstream promoter/enhancer activity due to insertion in active chromatin and preventing transcriptional interference—including enhancer activity attributed to the upstream piggyBac TR (Shi et al., 2007). We first generated a Tet response plasmid termed pB-TRE-Bi-EGFP (Figure 1B) expressing enhanced green fluorescent protein (EGFP). Next, we incorporated the transcription factor NEUROGENIN 2 (NEUROG2) that promotes glutamatergic-like neuron generation during cortical development (Zhang et al., 2013) and the DLX2 homeobox protein that promotes interneuron generation (Petryniak et al., 2007) into separate response plasmids to functionally test the ability of our system to inducibly direct differentiation. These two plasmids were defined as pB-TRE-Bi-EGFP/Ngn2 (i.e., Neurog2) and pB-TRE-Bi-EGFP/HA-Dlx2 (Dlx2 with a hemagglutinin [HA] epitope tag) (Figures 1C and 1D). For initial tests of the system, we transfected mouse N2a cells and a human cortical progenitor cell line (HuNPC) (Svendsen et al., 1998) with the three different response plasmids, along with rtTA-V10 and pBase. After culture for 4 days with or without dox, western blot analysis revealed robust GFP protein levels only in N2a cells grown with dox and no substantive GFP production without dox (Figure 1E). These collective results highlight both the inducibility and non-leakiness of the system. V5 epitope levels (from the TagBFP2-NLS in the response plasmid) demonstrated equivalent transfection and protein loading (Figure 1E). Moreover, NGN2 and HA (from the Dlx2 construct) were detected only in their respective groups when ICC analysis showed that HuNPCs nucleofected with pB-TRE-Bi-EGFP/HA-Dlx2 and grown with dox produced TagBFP2, GFP, and HA. In contrast, cells grown without dox produced only TagBFP2 (Figures 1F–1G1). Similarly, HuNPCs nucleofected with pB-TRE-Bi-EGFP constitutively expressed nuclear TagBFP2 but expressed GFP only with dox (Figures 1H and 1H1). Finally, NGN2 and GFP were induced only with dox in the pB-TRE-Bi-EGFP/Ngn2 group (Figures 1I–1J1). Interestingly, even though HuNPCs nucleofected with pB-TRE-Bi-EGFP are cortically derived, they did not express detectable NGN2 (Figures 1I1 and 1K1). Taken together, pB-Tet-GOI was readily inducible, non-leaky, and could be used to bidirectionally express a reporter and GOI simultaneously in vitro.