|SHEETS, TIMOTHY - University Of Maryland|
|TELUGU, BHANU - University Of Maryland|
Submitted to: Stem Cells and Development
Publication Type: Abstract Only
Publication Acceptance Date: 5/2/2015
Publication Date: N/A
Technical Abstract: Neurogenin 3 (NGN3) is a member of the basic helix-loop-helix transcription factor family. NGN3 is both necessary and sufficient to drive endocrine differentiation in the developing pancreas in mouse and humans. Until now, the sequence for NGN3 eluded discovery despite completion of the pig genome assembly. Using interspecies genetic synteny, sequence homology, and PCR, NGN3 was amplified from pig BAC genomic DNA, porcine genomic DNA and cDNA from pig pancreatic tissue. Sequencing data was uploaded to GeneBank. NGN3 gene was cloned into the pCR2.1 vector and subcloned into a constitutive lentiviral expression vector (FUGW). The long term objective is to address the dedifferentiation potential of non-endocrine pancreatic or liver cells into pancreatic endocrine hormone producing cells. The fidelity of the lentiviral vector expression was verified by transient transfection of 293ft cells and full length NGN3 protein verified by Western blot. Future experiments will test targeted ablation of NGN3 in primary porcine fetal fibroblasts via the CRISPR/Cas9 system. Cells will be co-transfected with a Cas9: GFP expression plasmid and a guide RNA vector targeting NGN3. Cells that are successfully transfected will be GFP-sorted into individual wells of a 96-well plate and clonally expanded. Colonies that are correctly targeted (as verified by sequence analysis of PCR amplified genomic fragments) will be selected for generation of NGN3-null pigs via somatic cell nuclear transfer. In conclusion, we believe we have identified a crucial pancreatic endocrine precursor gene, NGN3, whose conserved functionality will be tested using a domestic pig model.