Testing gene function early in the B cell lineage in mb1-cre mice - PubMed (original) (raw)
Testing gene function early in the B cell lineage in mb1-cre mice
E Hobeika et al. Proc Natl Acad Sci U S A. 2006.
Abstract
The mb1 gene encodes the Ig-alpha signaling subunit of the B cell antigen receptor and is expressed exclusively in B cells beginning at the very early pro-B cell stage in the bone marrow. We examine here the efficacy of the mb1 gene as a host locus for cre recombinase expression in B cells. We show that by integrating a humanized cre recombinase into the mb1 locus we obtain extraordinarily efficient recombination of loxP sites in the B cell lineage. The results from a variety of reporter genes including the splicing factor SRp20 and the DNA methylase Dnmt1 suggest that mb1-cre is probably the best model so far described for pan-B cell-specific cre expression. The availability of a mouse line with efficient cre-mediated recombination at an early developmental stage in the B lineage provides an opportunity to study the role of various genes specifically in B cell development and function.
Conflict of interest statement
Conflict of interest statement: No conflicts declared.
Figures
Fig. 1.
Targeting construct for the mb-1 locus with hCre recombinase. (a_–_c) mb-1 WT locus (a) and mb-1 locus (b) targeted by the mb1-cre before deletion of the neo cassette by the ACTB::Flpe mouse strain and after deletion of the neo cassette (c). The bar immediately upstream of the mb-1 promoter shows the approximate location of the probe used for the Southern blot analysis (shown in d) of genomic DNA isolated from WT and mb-1-targeted (mb1-hCre neo) ES cells. The endogenous mb-1 ATG in exon 1 was deleted. The FRT sites are represented by filled black arrowheads, and the tk neo cassette is represented by an open arrow. The mb-1 exons are shown as gray boxes numbered 1–5, and the EcoRI sites used for the Southern blot are labeled with R. The figure is not drawn to scale.
Fig. 2.
mb-1-cre activity is detected in the B lineage. Cells from bone marrow (bm), spleen (sp), lymph nodes (ln), and the peritoneal cavity (pc) from CD19-cre/Rosa-EYFP mice (a, c, e, and g) or mb-1-cre/Rosa-EYFP mice (b, d, f, and h) were stained for CD19 or IgM and analyzed by FACS. The percentage of CD19-positive B cells that were also EYFP-positive is depicted in the upper right quadrant of each FACS plot. All plots show cells in the lymphocyte gate. The plots are representative of at least three mice analyzed for each genotype.
Fig. 3.
Comparison of CD19-cre and mb1-cre activities in IL-7-dependent, bone marrow-derived pre-B cell cultures. Bone marrow-derived pre-B cells from CD19-cre/Rosa EYFP (a, c, and e) or mb1-cre/RosaEYFP (b, d, and f) mice were cultured for the indicated times in the presence of IL-7 and then analyzed by FACS. Cells were stained with anti-B220 antibodies. Similar results were obtained for two mice for each genotype.
Fig. 4.
mb1-cre/SRp20 shows a stronger B cell phenotype than CD19-cre/SRp20. (a) cre-mediated recombination of the targeted SRp20 locus (upper line) results in deletion of SRp20 exons 2 and 3. The loxP sites in introns 1 and 3 are indicated with solid arrows. (b) Single-cell suspensions from bone marrow (bm), spleen (sp), and peritoneal cavity (pc) were stained for B220, cKit, IgM, and IgD. The numbers in the plots indicate the percentages of total cells in the lymphocyte gate falling in each quadrant or region. For one mouse, a low number of B1 B cells was detected in the peritoneal cavity. DNA was isolated from sorted B cells derived from bone marrow, and spleen was PCR-amplified with primers specific for the floxed SRp20 locus. Bands corresponding to the floxed (fl), deleted (del), or WT alleles were resolved on agarose gels. Mice heterozygous (WT/fl), homozygous (fl/fl), or WT (wt/wt) for the floxed SRp20 gene either with (+) or without (−) the CD19-cre (c) or mb1-cre (d) gene were analyzed. The plots are representative of five mice analyzed for each genotype.
Fig. 5.
Deletion of Dnmt1 by mb1-cre results in a dramatic block in B cell development. (a) Schematic of the floxed Dnmt1 locus. Cre-mediated recombination deletes exons 4 and 5 of the Dnmt1 gene and a flanking hygromycin cassette introduced during construction of the floxed Dnmt1 strain. (b) B cells derived from the bone marrow (bm), peritoneal cavity (pc), and spleen (sp) of WT (Left), mb1-cre/Dnmt1 (Center), or mb1-deficient (Right) mice were analyzed by flow cytometry after staining for CD19, B220, cKit, IgM, and IgD. The plots are representative of at least three mice analyzed for each genotype.
Fig. 6.
Tissue specificity of mb1-cre recombination in floxed Dnmt1 mice. Recombination of the floxed Dnmt1 locus was accessed by Southern blotting using a probe derived from intron 2 of the Dnmt1 gene (see Materials and Methods). Genomic DNA from heart, kidney, thymus, liver, lung, bone marrow, and sorted splenic B cells was prepared from mice heterozygous for both the floxed Dnmt1 allele (WT/fl) and the targeted mb1-cre allele (WT/mb1-cre). As a control, genomic DNA derived from in vitro cultured bone marrow-derived pro/pre-B cells from mb1-cre/Dnmt1(fl/fl) bcl2 transgenic mice was also analyzed (lane 1).
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