RAG1 mutations affect V(D)J recombination in vivo. (A) Schematic of RAG1 showing the main core RAG1 subdomains. The positions of the mutations in the patient are shown with the equivalent mouse amino acids in brackets beneath. (B) Plasmid pJH299 used to quantify V(D)J recombination in vivo. Here, 12- and 23-RSSs are indicated as triangles. After inversional recombination, primers 1+2 and 3+4 are used in a qPCR nested assay, with a hydrolysis probe across the RSS junction. Total plasmid amounts are measured using primers 5 and 6. (C) Increasing amounts of WT RAG1 expression plasmid were transfected into NIH3T3 cells, and the levels of recombination were determined. A direct correlation with the amount of RAG1 activity is observed. n = 3; error bars show standard error of the mean (SEM). (D) Western blot showing expression levels of the various RAG1 proteins in transfected NIH3T3 cells; a β-galactosidase expression vector was co-transfected as a loading control. (E) Recombination levels using WT and mutated RAG1 proteins, relative to WT RAG1. Recombination was normalized according to the total amount of recombination plasmid recovered; similar levels of RAG1 protein expression were verified by western blotting (D). n = 3; error bars show SEM. CTD, carboxy-terminal domain; DDBD, dimerization and DNA binding domain; NBD, nonamer binding domain; preR, pre-RNaseH domain; ZBD, zinc binding domain.