Application of artificial endonucleases to the exocytotic mechanism of extracellular matrix proteins from periodontal ligament cells.

2016 Fiscal Year Final Research Report

• Project/Area Number: 26462822
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Functional basic dentistry
• Research Institution: Health Sciences University of Hokkaido
• Principal Investigator: Takuma Taishin 北海道医療大学, 歯学部, 名誉教授 (40095336)
• Project Period (FY): 2014-04-01 – 2017-03-31
• Keywords: ゲノム編集 / SNARE / 構成的分泌 / CRISPR/Cas9 / SNAP23 / 歯根膜細胞 / HAP1細胞 / TALEN

Outline of Final Research Achievements

In previous studies, constitutive exocytosis was normal in HeLa cells, whose SNAP23 was knocked down by up to 90% with its specific siRNAs, although SNAP23 null mice resulted in embryonic lethality at E3.5 before uterine implantation. To evaluate the role of SNAP23 in cell proliferation and constitutive exocytosis, we knocked out the gene by the application of CRISPR/Cas9 technology to HAP1 cells, a near haploid human cell line. The SNAP23-/ HAP1 cells had a 2-base pair-deletion in the SNAP23 gene; no SNAP23 protein was detectable by western blotting. The initial growth rate of the KO cell line was decreased by 40%, although the constitutive exocytosis of CLuc, a secreted luciferase of Cypridina noctiluca, and GFP-tagged human growth hormone was normal. The DNA microarray analysis showed that SNAP23 KO markedly influenced the expression of many genes. These results demonstrate that SNAP23 is not essential for cell proliferation or constitutive exocytosis.

Free Research Field

口腔生化学