FIG. 3.

Modification of cyclin A₁ protein levels resets the pluripotent state of iPS cells. (A) Western Blot analysis of cyclin A₁ protein levels in iPS clones with stable lentiviral shRNA for cyclin A₁, demonstrating 50% knockdown of protein expression. Graph showing quantification of cyclin A₁ protein levels after shRNA treatment. An FAC analysis of a cell cycle profile of miPS cell clones compared to treated with stable lentiviral shRNA for cyclin A₁. (B) Table summarizing presence of green fluorescent protein (GFP) in gonads or the embryo body of chimera mice made from GFP iPS cells or GFP iPS cells treated with RNAi for cyclin A₁. Below are photographs of gonads (with control gonads, CG, of a GFP-OCT4 mouse) and the embryo body at d12.5 showing GFP-cyclin A₁RNAi signal. (C) A functional pluripotency assay was performed by making chimera mice; the table summarizes the percentage of chimeras and the percent contribution to coat color of chimera mice for iPS cells with and without cyclin A₁ knockdown. A total of 331 embryo transfers were made for iPS cell lines, and 300 embryo transfers were performed for iPS cell lines treated with cyclin A₁ knockdown. Photographs showing example of chimera litter for each condition summarized in the table. Color images available online at www.liebertonline.com/scd