Figure 1.

A new reporter cell line for purification of 2C-like cells. (A) Reporter design: A previous MERVL-GFP reporter (Ishiuchi et al. 2015) was modified to contain the extracellular portion of the CD4 antigen downstream from GFP and a T2A cleavage element, allowing rapid 2C-like cell purification by anti-CD4 beads. (B) Representative flow cytometry plot depicting proportion of typical 2C-GFP⁺ enriched cells (>60% pure, 2C-pos) cells before (left) and after (right) CD4-based 2C enrichment. (C) Percent recovery of 2C-GFP-pos cells after CD4-based purification, comparing CD4⁺ cells (eluate) and CD4⁻ cells (flowthrough). Data are mean ± SEM of three experiments. (D) qRT-PCR validation of high levels of 2C-specific genes and MERVL in the 2C-pos, CD4⁺ eluate compared with 2C-neg, CD4⁻ fraction and the starting population. Flowthrough cells are set to 1. Data are mean ± SEM of three experiments. (E) Representative confocal images and (F) quantification of levels of Oct4 and MERVL gag proteins and DAPI in 2C-pos versus 2C-neg cells following CD4-based purification. Scale bar, 20 µm. All P-values represent two-tailed, unpaired Student's t-test, with multiple comparisons correction where relevant.