Expression profiling by high throughput sequencing
Summary
The goals of this study are to compare the transcriptomes of control heterozygous and Tuberous sclerosis 2 (Tsc2) homozygous nociceptors 3 days after sciatic nerve injury that have been enriched by fluorescence-associated cell sorting (FACS) with the aim of identifying differences in genes associated with axon regeneration. Nav1.8-Cre transgenic mice were used to delete Tsc2 as well as express Green fluorescent protein from the Rosa26 locus. Tsc2 deletion constitutively activates mTORC1 signaling cell autonomously. Dissociated dorsal root ganglia from adult mice were FACS-sorted for GFP and analyzed by RNA-seq. We find that the expression of a number of regeneration-associated transcription factors including cJun, Atf3, Sox11 and other are upregulated in Tsc2 mutant neurons in the absence of an injury. Additionally, the target genes of several other normally expressed regeneration-associated transcription factors are differentially expressed in Tsc2 mutant neurons, such as Creb1 and Klf’s. We conclude Tsc2 deletion and consequent mTORC1 activation establishes a pro-regenerative gene expression profile in nociceptors in the absence of an injury.
Overall design
L4 dorsal root ganglia contralateral and ipsilateral to sciatic nerve crush were harvested 3 days post-injury from mice with Nav1.8-Cre mediated expression of GFP from Rosa26 locus as well as heterozgous (control) or homozygous (cKO) deletion of Tsc2 were FACS-sorted by GFP expression for 3 technical replicates of 100 cells each. RNA-seq data from 4 biological replicates was collected.
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