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. 2015 Sep 30;35(21):3701–3713. doi: 10.1128/MCB.00219-15

FIG 5.

FIG 5

A pair of RSSs improves RAG recruitment efficiency. (A) The p12/23 retroviral recombination substrate. The 5′ and 3′ long terminal repeats (LTR), the mouse CD90 gene encoding Thy1.1 that lies in the opposite transcriptional orientation, the human CD4 (hCD4) gene, the internal ribosome entry site (IRES), and the 12RSS (12) and the 23RSS (23) are indicated (25). (B) Simplified schematic of p12/23 and its variants pNone (lacking the 12RSS and 23RSS), p12 (lacking the 23RSS), and p23 (lacking the 12RSS). Primer pairs 12a (gray underlining) and 23d (black underlining) span the locations of the 12RSS and 23RSS, respectively (25). (C and D) Binding of RAG1 (C) and RAG2 (D) in the D345 cell line over a 48-h time course of STI induction at the 12RSS and 23RSS sites was assessed by ChIP-qPCR. Insets show the RAG1 data for the 12RSS site of p12 and the 23RSS site of p23, presented with the same y axis scale as that for Jκ gene segments in Fig. 3B. (E) H3K4me3 levels at each RSS site for each substrate. Bars and dot plots indicate the means of data from ≥3 independent experiments, and error bars represent the standard errors of the means.