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. 2009 Jan 19;206(1):153–167. doi: 10.1084/jem.20081202

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© 2009 Li and Eckhardt

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jem.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

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Figure 5. — V-DJ rearrangements on the WT Igh^b allele of V_HEμ^a/WT^b and V_HΔ^a/WT^b mice. (A) Splenic B cells from WT^a/WT^b and V_HΔ^a/WT^b, sorted on the basis of phenotype (IgM^a+b− or IgM^a+b+). PCRs were performed on fivefold serial dilutions of DNA templates (four lanes/cell type). PCR strategy detects V-DJ rearrangements on only the Igh^b allele in V_HΔ^a/WT^b mice (both alleles in WT^a/WT^b mice). VDJ rearrangements involving each J_H gene segment are indicated (J_H1, J_H2, etc.). V_H family primers are indicated to the right of blots (V_HJ558, etc.). PCR of HS4 (a 3′ RR element) was normalized for DNA input. (B) Like A, but a primer designed to anneal to all V_H families (promiscuous VH 46) was included. (C) D-J rearrangements on the Igh^b allele of pre-B cells (B220⁺CD43⁻IgM⁻) sorted from six mice of each genotype. The D_HL primer anneals to most D_H genes (47); DQ52 anneals only to D_HQ52. Allele-specific 3′ primer (J_H4) was used as in A. Liver DNA from WT^a/WT^b mice and spleen cell DNA from homozygous V_HEμ^a and V_HΔ^a mice were included as controls. H₂O, no template control. (D) Pre-B cells were isolated as in C and PCR reactions using the V_H gene family primers were performed as described in A. (E) Pre-B cells were isolated as in C. Sample dilutions determined to be in the linear range (Fig. S4, available at http://www.jem.org/cgi/content/full/jem.20081202/DC1) were prepared in triplicate for PCR. Representative data is shown. (F) Quantitative analysis of VDJ rearrangement blots. For each PCR reaction with a given V_H primer, signals for J_H1, J_H2, J_H3, and J_H4 rearrangements were quantified and summed by ImageQuant. Values obtained for pre-B cells from V_HEμ^a/WT^b and V_HΔ^a/WT^b mice were normalized to the corresponding values for pre-B cells of WT^b/WT^b mice. Data were obtained from two pools of two mice each for WT^b/WT^b and three pools involving a total of seven mice for each of the V_HEμ^a/WT^b and V_HΔ^a/WT^b mouse lines. Error bars show SD.