B cell development past the pro-B cell stage in mice requires the Cul4-DDB1-Roc1 E3 ubiquitin ligase substrate recognition subunit VprBP. in B cells impairs selection of Igκ “editor” light chains typically arising through secondary rearrangement but not selection of Igλ editor light chains. Both heavy and light chain site-directed transgenic mice show increased B cell anergy when VprBP is inactivated in B cells. Taken together these data argue that VprBP is required for the efficient receptor editing and selection of Igκ+ B cells but is largely dispensable for Igλ+ B cell Imipramine Hydrochloride development and selection and that VprBP is necessary to rescue autoreactive B cells from anergy induction. early in B cell development arrests B cell maturation at the pro B-to-pre-B cell transition but this developmental block is partially rescued by expressing functionally rearranged Ig transgenes. Loss of VprBP expression in B cells is associated with impaired VH-DJH gene rearrangement reduced fidelity of VH-DJH joining defects in cell cycle progression and increased apoptosis (3). Given the elevated levels of apoptosis observed in VprBP-deficient B cells here we investigated whether enforced expression of the pro-survival factor Bcl2 can compensate for the loss of VprBP during B cell development as has been observed in other cases of genetic insufficiency manifesting impaired B cell development (4-7). As Imipramine Hydrochloride in those cases we find that expression partially rescues B cell development substantially Imipramine Hydrochloride reconstituting marginal zone but not follicular B cell populations. Unexpectedly however most B cells maturing under this program express Igλ rather than Igκ. The loss of Igκ+ B cells in this context can be partially rescued in mice bearing a site-directed Igκ light chain transgene suggesting VprBP does not regulate light chain expression from a productively rearranged allele. More detailed analysis of V(D)J rearrangement patterns in pre-B cells and rare Igκ+ B cells isolated from VprBP-deficient mice provides evidence for inefficient distal VH-DJH gene rearrangement and secondary rearrangements associated with receptor editing in these animals. However the apparent V(D)J recombination defects are substantially rescued by enforced Bcl2 expression ruling out a direct role for VprBP in mediating the V(D)J rearrangement process itself. As an alternative we speculated that VprBP functions indirectly to regulate the efficiency of B cell receptor editing and selection of Igκ+ B cells. To test this possibility we analyzed how the loss of VprBP function affects B cell development and selection in mice harboring the site-directed VH3H9/56R (56R) anti-DNA heavy chain transgene which is Imipramine Hydrochloride used as a model of VH gene replacement as well Imipramine Hydrochloride as light chain receptor editing and selection (8). Our results suggest that VprBP insufficiency impairs VH gene replacement and selection of Igκ editor light chains but does not interfere with the selection of Igλ editor light chains. Interestingly both heavy and light chain site-directed transgenic mice show an increased frequency of phenotypically anergic B cells when VprBP is inactivated. Taken together these data argue that VprBP is required for the efficient editing and selection of Igκ+ B cells but is largely dispensable for Igλ+ B cell development and selection and is necessary to salvage B cells from potential anergy induction. Materials and Methods Mice Mice with the following conditional alleles or transgenes have been previously described: and IRS-RS rearrangements were amplified by PCR from template DNA (10000 2500 and 625 genome-equivalents). Briefly PCR reactions Imipramine Hydrochloride (50 μl) containing template DNA and 0.5 μM of each VPS15 primer (see Table 1) in sample buffer (0.2 mM of dNTPs 20 mM Tris-HCl (pH 8.4) 50 mM KCl 1.5 MgCl2 and 2.5 units Taq polymerase [Promega Madison WI]) were subjected to initial denaturation (and IRS-RS rearrangements: 94°C for 30 sec 59 for 1 min 72 for 2 min; IgVλx rearrangements: 94°C for 20 sec 60 for 30 sec 72 for 1.5 min; IgλR1 rearrangements: 94°C for 30 sec 48 for 1 min 72 for 2 min; Vκ1 rearrangements: 94°C for 30 sec 60 for 1 min 72 for 2 min; Vκ21 rearrangements: 94°C for 30 sec 55 for 1 min 72 for 2 min) and then a final extension (approach to conditionally disrupt expression in the B.