Figure 6.

Long-range PCR identified class switch deletions in C_H1. DNA preparations from one total spleen and sorted syn⁺ spleen cells from four L^−/− mice were analyzed. (A) PCR amplifications (lanes a–f) from DJ_H to γC_H2^e (40 cycles), using a primer (VDJ029) based on the H chain sequences obtained by RT-PCR (left) or from J_H4long to γC_H2^e (20 cycles; right). The thin white line indicates where the original gel was spliced. In the reactions, cell aliquots from one (single) and three (pool) L^−/− mice were used. (B) Nested PCR (28 cycles) of first-round products (lanes a–f) from Eμ to γC_H2^d, with cloned products indicated by arrows. Controls were a γ2a hybridoma (hybrid), ES cell DNA, and amplification without DNA (−). (C) Map of the amplified genomic region from J_H4 to Cγ exons C_H1, hinge (H), and C_H2. Cloning and sequencing of PCR products showed deletions of large parts of the switch region and some or all of C_H1. Clone numbers (left) and sizes (right), from 3′ Eμ to γC_H2^d, are indicated, and sequences are compiled in Fig. S1.