Cre recombinase-controlled expression of the mb-1 allele.

Abstract

The mb-1 gene is located on chromosome 7 of the mouse genome and it is composed of five exons encoding Ig, which is a B cell-specific protein and an element of the B cell antigen receptor complex (BCR) (Sakaguchi et al., 1998; Hombach et al., 1990). Expression of Igis required for the transport of BCR onto the cell surface. Together with Ig, Igforms a heterodimer that mediates signal transduction from the BCR (Kim et al., 1993). The mb-1 gene is expressed at every stage of B cell development and activation, excluding terminally differentiated plasma cells. The knock-out of mb-1 results in complete block of B cell development at the progenitor stage in mice, as well as in humans (Minegishi et al., 1999; Pelanda et al. unpublished observations). Thus, we created conditional alleles of mb-1 to study the behavior of mature B cells with or without Igexpression. Cre-mediated recombination of inverted loxP sites results in the inversion of the loxP-flanked (floxed) DNA sequence (Nagy 2000). By flanking the mb-1 coding sequence with inverted loxP sites we generated a conditional mb-1 allele that allows switching “on” or “off” (and vice versa) of mb-1 expression under the control of Cre recombinase. In addition, BAP31N-EGFP, which encodes a membrane-associated form of the enhanced green fluorescent protein (mEGFP), was cloned in the opposite transcriptional orientation relative to mb-1 coding sequence into the conditional mb-1 allele. This allows the detection of cells at the “off” state of mb-1 transcription by the expression of mEGFP. Embryonic stem (ES) cells carrying the conditional mb-1 allele were generated by homologous recombination following standard techniques (Torres and Kuhn 1997). By transiently transfecting these latter cells with a Cre-expressing vector, we isolated ES clones carrying the deletion of the neomycin resistance (neo) cassette and the floxed mb-1 targeted allele in either orientation, thus resulting in the generation of mice carrying either mEGFP/mb-1 or mb-1/mEGFP conditional alleles. A schematic representation and Southern analysis of wild-type and targeted mb-1 alleles are shown in Figure 1a–c. Expression of mEGFP (Fig. 2) and of Ig(data not shown) by the targeted mb-1 alleles was confirmed by flow cytometric analysis of bone marrow, spleen, and lymph node cells isolated from heterozygous mEGFP/mb-1 and homozygous mb-1/mEGFP mice, respectively. The mEGFP/mb-1 allele directs expression of mEGFP, but not Ig. Thus we found that homozygous mEGFP/mb-1 mice lack any Igexpression and their B cell development is absolutely blocked at the B cell progenitor stage (data not shown). On the other hand, the mb-1/mEGFP allele encodes for Ig, but not mEGFP (Fig. 2c and data not shown). The expression of Igby the conditional allele was found to be reduced compared to wild-type (data not shown). As expected and observed in previous cell line experiments, the continuous presence of Cre in the nuclei results in the repetitive inversion (flipping) of the floxed DNA sequence that carries inverted loxP sites (data not shown). Removal of nuclear Cre stops further loxP recombination, stabilizing the floxed DNA into one of the two possible orientations with equal probability. Thus, the resulting cell population contains cells with either orientation of the floxed DNA sequence. The conditional mb-1 mice were tested by breeding them to either polyI/C, (MxCre, Kuhn et al., 1995) or hydroxy-tamoxifen, (MerCreMer, Hobeika et al., unpublished) inducible Cre-transgenic mouse strains. Induction of Cre recombinase in mEGFP/mb-1;MxCre heterozygous mice resulted in the elimination of mEGFP expression in approximately 30% of the bone marrow and splenic B cells (Fig. 2a). Southern blot analysis confirmed the presence of cells carrying mb-1/mEGFP alleles, which were the result of Cre-mediated recombination of mEGFP/mb-1 alleles (Fig. 2b). On the other hand, Cre induction in mb-1/mEGFP;MerCreMer bone marrow cells in vitro resulted in the de novo expression of mEGFP in approximately 9% of B cells (Fig. 2c). Thus, our experiments demonstrated that the mb-1 targeted alleles described here are functional with respect to their capacity to be conditionally activated by inducible Cre recombinase. Conditional mb-1 mice will allow study of the kinetics and functions of B cell development by the induction of Igexpression and the role of Igand BCR signaling in mature B cells, by suppression of Igexpression.