Figure 4. Visualization of Eμ-dependent locus contraction.

(A) The unrearranged IgH locus as represented in Figure 2A showing the location of six 10 kb probes used for FISH.

(B) Three color 3D-FISH were carried out with bone marrow pro-B cells of the indicated IgH genotypes cells in a RAG2-deficient background. Short probes labeled with Alexa Fluor 594 (red) and 488 (green), and BAC RP23-201H14 labeled with Alexa Fluor 697 (blue) were hybridized to fixed pro-B cells. Signals were visualized by epifluorescence microscopy and distances between probes were determined as described (Jhunjhunwala et al., 2008) and shown in Table S1. Probe combinations were: a, d, g h4-red, h11-green; b, e, h h4-red, 5′7183-green; c, f, i h4-green, 3′558-red. Red line represents 1 μm.

(C) Quantitation of FISH data shown in part B. Distances between red and green 3D FISH signals in part A were divided into 5 categories (<0.2, 0.2-0.5, 0.5-0.8, 0.8-1.0, and >1.0 μm) for 60-90 nuclei. The percentage of IgH alleles in each category was determined (Y axis) for each IgH genotype (X axis) and is represented in different colors. Probe combinations are shown above the bars. Pro-B cells were purified from 5-6 mice of each genotype.

(D) Three color 3D-FISH with RAG2-deficient pro-B cell lines carrying WT and Eμ- deleted IgH alleles. Probe combinations were: a, c h4-red, h11-green; b, d h4-green, 3′558-red.

(E) Quantitation of the FISH data shown in part D as described in C.

(F) Three color 3D-FISH with bone marrow pro-B cells of the indicated IgH genotypes cells obtained from RAG2-deficient background. Probe combinations were: a, c h1-red, h11-green; b, d DFL-red, 3′RR-green. Pro-B cells were purified from 5-6 mice of each genotype.

(G) Quantitation of FISH data shown in part F as described in C.