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Research reveals that the wild-type and mutant zinc finger peptides bind to their respective binding sequences. However, the N-terminal finger of the mutant peptides exhibit varying degrees of base recognition due to the C-terminal finger induced conformational changes in the DNA.

Research was conducted to study the DNA recognition mode of human transcription factor Sp1 by examining the influence of zinc finger 1 to the high-affinity binding to the GC box DNA. Oligonucleotides were annealed and inserted between a BamHI site and an EcoRI site while mutated GC box sequences were utilized to analyze the DNA sequence selectivity of finger 1. Results showed that the interaction mechanism between finger 1 and DNA differs from the Zif268 model.

Purpose: To establish a practical research tool for studying the pathogenesis of retinal ganglion cell (RGC) diseases, we optimized culture procedures to induce neurite outgrowth from three-dimensional self-organizing optic vesicles (3D-retinas) differentiatedin vitrofrom mouse and human embryonic stem cells (ESCs). Materials and methods: The developing 3D-retinas isolated at various time points were placed on Matrigel-coated plates and cultured in media on the basis of the 3D-retinal culture or the retinal organotypic culture protocol. The number, length, and morphology of the neurites in each culture condition were compared. Results: First, we confirmed that Venus-positive cells were double-labeled with a RGC marker, Brn3a, in the 3D-retina differentiated from Fstl4::Venus mouse ESCs, indicating specific RGC-subtype differentiation. Second, Venus-positive neurites grown from these RGC subsets were positive for beta-III tubulin and SMI312 by immunohistochemistry. Enhanced neurite outgrowth was observed in the B27-supplemented Neurobasal-A medium on Matrigel-coated plates from the optic vesicles isolated after 14 days of differentiation from mouse ESCs. For the differentiated RGCs from human ESCs, we obtained neurite extension of >4 mm by modifying Matrigel coating and the culture medium from the mouse RGC culture. Conclusion: We successfully optimized the culture conditions to enhance lengthy and high-frequency neurite outgrowth in mouse and human models. The procedure would be useful for not only developmental studies of RGCs, including maintenance and projection, but also clinical, pathological, and pharmacological studies of human RGC diseases. [ABSTRACT FROM PUBLISHER]