Major histocompatibility class II (MHC-II) molecular complexes bind antigenic peptides and present them to antigen recognition receptors, and as such are of fundamental importance to the adaptive immune system of higher organisms (Janeway et al., 1999). Inactivation of the MHC-II beta chain (IAb) gene results in loss of cell surface MHC-II expression (Cosgrove et al., 1991). We describe the generation of a conditional IAb (iabneo) null allele that, when combined with appropriate Cre recombinase transgenic mice, should allow us to dissect the MHC-II-dependent immune functions of discrete subsets of dendritic cells. For example, it has been proposed that the CD8a-positive subset of dendritic cells normally resident in secondary lymphoid organs might have regulatory roles with respect to T-cell reactivity (Suss and Shortman, 1996), but the in vivo significance of this is not yet clear. The ability to specifically ablate MHC-II from CD8a-positive dendritic cells would allow us to address this and other issues. This iabneo allele still contains a loxP-flanked neomycinresistance cassette (Fig. 1), but homozygous iabneo/neo mice appear to have relatively normal levels of MHC-II on B cells and dendritic cells (data not shown but see Fig. 2). To determine whether the iabneo allele can be deleted with resultant loss of cell surface MHC-II, we employed our TATCre reagent. The latter is a cell-permeable Cre-recombinase protein described in detail elsewhere (Joshi SK, Hashimoto K, Koni PA, submitted). Briefly, we have generated a TAT transporter-Cre-recombinase fusion protein (referred to as TATCre) that is capable of transducing into cells and then mediating DNA recombination vialoxP sites, a phenomenon that we refer to as DNA-recombinationafter-transduction (DRAT) activity. TATCre was created to allow Cre-recombinase-mediated deletion without longterm Cre-recombinase exposure, as a number of recent studies have shown that Cre recombinase can cause toxicity in the form of chromosomal aberrations, for example, if overexpressed. TATCre protein was prepared using the basic principles described by others (Nagahara etal., 1998) and described in detail elsewhere (Joshi SK, Hashimoto K, Koni PA, submitted).

A single treatment of splenocytes from iabneo/neo mice with TATCre as in Figure 2 resulted in loss of cell surface MHC-II protein from about 80% of B220-positive B cells. The expression of MHC-II on B cells from C57BL/6J mice was not affected by TATCre treatment (Fig. 2). Two treatments with TATCre caused greater than 99% deletion from B220-positive B cells (Fig. 2) but was associated with increased cell death as judged by Trypan-blue staining (data not shown) and reduced overall live cell recovery (Fig. 2). The B220-positive B cells represented about 85% of the live cells in these cultures and there were no other detectable MHC-II-positive cells (data not shown).