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. Author manuscript; available in PMC: 2015 Oct 30.
Published in final edited form as: J Immunol. 1995 Jan 1;154(1):226–238.

FIGURE 2.

FIGURE 2

Construction of the recombinant leader-V(D)J13 gene segment (leader-VHDJH13 or leader-VκJκ13) (A), of the recombinant “leader-Vgerm-line(D)J13” gene segment (B), and of the recombinant gene segment encoding the mutant “Gly31” (C). Open boxes depict restriction sites. Broken arrows indicate nucleotide chain elongation by DNA polymerase. Solid ( Inline graphic), hatched ( Inline graphic), and shaded ( Inline graphic) segments depict the sequences of the expression vectors (pcDNAIG or pSXRDIG), that of the V(D)J gene segment of mAb13, and those of autologous germ-line genes, respectively. To construct the recombinant leader-V(D)J13 gene segment (leader-VHDJH13 or leader-VκJκ13), the mouse leader Ig V gene segment in the expression vector and the VHDJH or VκJκ gene segment of mAb13 were amplified in separate PCR (PCR 1 and PCR 2), and joined by recombinant PCR. Primers (Ov1 and Ov2) used at the end to be joined were made complementary to one another by including nucleotides at the 5′ end that are complementary to the 3′ portion of the other primer (see Table I). Primers, leader, and FR4 were designed to yield final recombinant products bearing HindIII and XhoI sites used for the introduction into the expression vectors. To construct the recombinant leader-Vgerm-line(D)J13 gene segment, the gene segment amplified from the autologous germ-line gene using the sense Ov2 and antisense FR3 B primers (GIII-FR3 B or κIII-FR3 B) (PCR 1) and that amplified from the mAb13 V(D)J gene using the sense FR3 A (GIII-FR3 A or κIII-FR3 A) and antisense FR4 (PCR 2) were joined by recombinant PCR. This recombinant gene segment was fused to the mouse Ig leader sequence (amplified by PCR 3) by recombinant PCR. To construct the recombinant gene segment encoding the mutant “Gly31”, the DNA segment amplified from the leader-VHDJH segment using the leader and 13-FR2 B primers (PCR 1) and that amplified from the recombinant leader-VH13G12-DJH13 segment using the 13-FR2 A and FR4 primers (PCR 2) were joined and amplified by recombinant PCR. See Table I for sequences of the sense 13-FR2 A and the antisense 13-FR2 B primers.