(?): = 0

(?): = 0.00031, = 11.63, degrees of freedom = 4, 95% CIdifference = 129,913 to 211,436. in (23) but also with some studies in cultured mammalian cells (25C27) and embryos (26). In the mean time, calling cards has also been successfully L 006235 applied to yeast (28) and mammalian cell (18) model systems. However, neither of these methodologies has to date been implemented for TF recording in postnatal mammalian model systems, such as mice. Here, we adapt calling cards for in vivo use by delivering this system to the mouse brain via adeno-associated computer virus (AAV). This method, in the mold of traditional calling cards technologies (18), works by first expressing the (hypPB) transposase within a cell and providing donor transposons. hypPB inserts donor transposons at TTAA sites throughout the genome, leaving permanent marks, or calling cards, at these loci. These transposons can later be sequenced and mapped to the genome to record the history of hypPB localization across the genome. hypPB-mediated insertions can be used to assess TF binding in two ways: 1) hypPB may be fused to a TF of interest, so that the TF directs the insertion of transposons near its genomic binding sites (18); or 2) unfused hypPB directly interacts with the bromodomain and extraterminal domain name (BET) protein, BRD4, and directs transposon DNA into BRD4-associated genomic regions (29, 30), most prominently active super enhancers (7). We establish that calling cards systems can be delivered to the mouse brain via AAV and that these components successfully record TF occupancy without the need for any TF-specific antibody. We then produce a conditionally expressed, Cre recombinase-dependent version of AAV calling cards, termed FlipCExcision, or FLEX, calling cards and demonstrate, as a proof of theory, the ability of this system to record cell type-specific TF-occupancy profiles in the brain. Lastly, we provide evidence that through continued transposon insertion, FLEX calling cards can record and integrate TF-binding events over extended time periods following viral delivery, providing insights into transient TF activity that would be normally missed with end-point steps such as ChIP-seq. Results Intracranial Delivery of Calling Cards via AAV Invokes Common Transposon Insertion in the Mouse Cortex. In order to perform transposon calling cards in mammalian cells, two basic components are required: the hypPB transposase (or a TFChypPB fusion) and donor transposons (18). We sought to develop an in vivo method to efficiently deliver calling cards L 006235 components throughout the mouse brain to identify TF-associated sites. We first tested AAV as a means for calling cards reagent delivery, as viral delivery methods have been successful in other organ systems previously (31, 32). We packaged a myc-tagged version of hypPB and donor transposons transporting TdTomato reporter genes into individual AAV serotype 9 (AAV9) vectors, which efficiently transduce neuron and astrocyte populations (33), and intracranially injected these vectors into the cortices of postnatal day 0 to 1 1 (P0-1) mice. Animals were killed at P21 for analysis (Fig. 1and and = Rabbit Polyclonal to MYT1 1,005 myc(+) cells, counted across cortical image fields from 5 mice. (test, > 0.05; n.s., not significant). (and = 21) or control, RFP-only (= 24) viruses displayed no significant differences in anxiety-related behavior (center/edge dwelling) (test, with Bonferroni-corrected = 0.05 as a significance threshold (including all assessments in and and and and and and and < 10?30) displaying high correlation between replicates (= 0.994). (axis represents the number of reads supporting each insertion on a log10 level, and the bottom track L 006235 displays normalized local insertion density across the genome L 006235 (insertions per million per L 006235 kilobase [kB]). The axis of ChIP-seq data represents read depth with smoothing filter applied. Warmth maps and enrichment plots are centered on insertion.