Development of versatile non-homologous end joining-based knock-in module for genome editing

CRISPR/Cas9-based genome editing has dramatically accelerated genome engineering. An important aspect of genome engineering is efficient knock-in technology. For improved knock-in efficiency, the non-homologous end joining (NHEJ) repair pathway has been used over the homology-dependent repair pathway, but there remains a need to reduce the complexity of the preparation of donor vectors. We developed the versatile NHEJ-based knock-in module for genome editing (VIKING). Using the consensus sequence of the time-honored pUC vector to cut donor vectors, any vector with a pUC backbone could be used as the donor vector without customization. Conditions required to minimize random integration rates of the donor vector were also investigated. We attempted to isolate null lines of the VDR gene in human HaCaT keratinocytes using knock-in/knock-out with a selection marker cassette, and found 75% of clones isolated were successfully knocked-in. Although HaCaT cells have hypotetraploid genome composition, the results suggest multiple clones have VDR null phenotypes. VIKING modules enabled highly efficient knock-in of any vectors harboring pUC vectors. Users now can insert various existing vectors into an arbitrary locus in the genome. VIKING will contribute to low-cost genome engineering

Mast Cells and Angiogenesis in Oral Malignant and Premalignant Lesions

Mast cell contribution to neoangiogenesis during tumorigenesis in oral squamous cell carcinoma is not determined yet. Objectives: To associate numerical mast cell density (MCD) to numerical microvessel density (MVD) during the progression of oral leukoplakia without dysplasia and leukoplakia with dysplasia to squamous cell carcinoma (OSCC). Materials and methods: MVD was analysed immunohistochemically (mouse monoclonal anti-human CD34) in 49 paraffin-embedded specimens, 35 OSCCs, 9 leukoplakias and 5 normal oral tissues. Toluidine blue counterstaining revealed mast cells. MCD and MVD were assessed at the same optical field. Results: MVD increased between: normal oral mucosa, dysplasia (p=0.004), OSCC (p=0.001), leukoplakia and OSCC (p=0.041). MCD increased between: normal oral mucosa, dysplasia (p=0.003), OSCC (p=0.000), leukoplakia and OSCC (p=0.007). MVD was found to depend on MCD (p=0.000) in a percent 28.3% (power curve fit model). Conclusions: Mast cells are attracted at the lesion site and may turn on an angiogenic switch during tumorigenesis in OSCC

An NRSF/REST-like repressor downstream of Ebi/SMRTER/Su(H) regulates eye development in Drosophila

The corepressor complex that includes Ebi and SMRTER is a target of epidermal growth factor (EGF) and Notch signaling pathways and regulates Delta (Dl)-mediated induction of support cells adjacent to photoreceptor neurons of the Drosophila eye. We describe a mechanism by which the Ebi/SMRTER corepressor complex maintains Dl expression. We identified a gene, charlatan (chn), which encodes a C2H2-type zinc-finger protein resembling human neuronal restricted silencing factor/repressor element RE-1 silencing transcription factor (NRSF/REST). The Ebi/SMRTER corepressor complex represses chn transcription by competing with the activation complex that includes the Notch intracellular domain (NICD). Chn represses Dl expression and is critical for the initiation of eye development. Thus, under EGF signaling, double negative regulation mediated by the Ebi/SMRTER corepressor complex and an NRSF/REST-like factor, Chn, maintains inductive activity in developing photoreceptor cells by promoting Dl expression