Precise control of cell proliferation and differentiation is critical for organogenesis. Geminin (Gem) has been proposed to link cell cycle exit and differentiation as a prodifferentiation factor and plays a role in neural cell fate acquisition. Here, we identified the SWI/SNF chromatin-remodeling protein Brg1 as an interacting partner of Gem. Brg1 has been implicated in cell cycle withdrawal and cellular differentiation. Surprisingly, we discovered that Gem antagonizes Brg1 activity during neurogenesis to maintain the undifferentiated cell state. Down-regulation of Gem expression normally precedes neuronal differentiation, and gain-and loss-of-function experiments in Xenopus embryos and mouse P19 cells demonstrated that Gem was essential to prevent premature neurogenesis. Misexpression of Gem also suppressed ectopic neurogenesis driven by Ngn and NeuroD. Gem's activity to block differentiation depended upon its ability to bind Brg1 and could be mediated by Gem's inhibition of proneural basic helix-loop-helix (bHLH)-Brg1 interactions required for bHLH target gene activation. Our data demonstrate a novel mechanism of Gem activity, through regulation of SWI/SNF chromatin-remodeling proteins, and indicate that Gem is an essential regulator of neurogenesis that can control the timing of neural progenitor differentiation and maintain the undifferentiated cell state.[Keywords: Brg1; Geminin; neurogenesis; proneural bHLH; SWI/SNF chromatin-remodeling complex; Xenopus] Supplemental material is available at http://www.genesdev.org.

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The LEAP (Laser‐Enabled Analysis and Processing) platform combines in situ imaging with laser manipulation to efficiently identify, purify, and monitor expansion of high secreting clones. It also allows for rapid analysis of cell population heterogeneity. This unit describes the LEAP instrumentation as well as basic and alternate protocols of the major applications in recombinant human or humanized IgG expression. The protocols include fluorescent cell counting, secreted recombinant IgG capture and detection, and IgG‐secreting clone selection by laser processing. Curr. Protocol. Cytom. 43:2.14.1‐2.14.27. © 2008 by John Wiley & Sons, Inc.

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