Volume 7, Issue 6, Pages 765-775 (December 1997) Receptor Editing in a Transgenic Mouse Model: Site, Efficiency, and Role in B Cell Tolerance and Antibody Diversification  Roberta Pelanda, Stephan Schwers, Eiichiro Sonoda, Raul M Torres, David Nemazee, Klaus Rajewsky  Immunity  Volume 7, Issue 6, Pages 765-775 (December 1997) DOI: 10.1016/S1074-7613(00)80395-7

Figure 1 Targeted Insertions of V Region Genes and Model for the In Vivo Analysis of Receptor Editing (A) Schematic representations of the IgH and Igκ loci carrying the insertion of, respectively, the 3-83H and B1-8H genes, and the 3-83κ gene. The two independent targeting approaches of the 3-83κ gene are shown. Empty rectangles and circles represent VH, Vκ, DH, Jκ and constant regions and specific enhancers. Black triangles located upstream of the Ig gene insertions (marked rectangles) represent residual lox P sites. (B) Schematic representation of the Ig loci that encode nonautoreactive and autoreactive receptors in the present model system. Open or filled rectangles, V, D, and J regions; open circles, enhancers; filled, arrowed box, the RS sequence. Immunity 1997 7, 765-775DOI: (10.1016/S1074-7613(00)80395-7)

Figure 2 Flow Cytometric Analysis of Idiotype Expression in Spleen Cells from Igi Mice Splenocytes isolated from adult B1-8Hi;3-83κi, B1-8Hi, 3-83Hi;3-83κi, and 3-83Hi mice were stained with anti-CD45R/B220, anti-IgM, and anti-idiotypic monoclonal antibodies and analyzed by flow cytometry. Anti-idiotypic antibodies were, respectively, Ac-146 (anti-B1-8) or 54.1 (anti-3-83). The figure shows the analysis of anti-IgM and anti-idiotype staining of cells in the lymphocyte gate. Numbers indicate the percentage of idiotype-positive splenocytes within the IgM+ cell population. Immunity 1997 7, 765-775DOI: (10.1016/S1074-7613(00)80395-7)

Figure 5 Three-Color Flow Cytometric Analysis of Bone Marrow Cells from Wild-Type B1-8Hi;3-83κi and 3-83Hi;3-83κi Mice Bone marrow cells were analyzed for the expression of CD45R/B220, IgM, and CD43/S7. (Top) B220 versus IgM expression of cells in the lymphocyte gate. (Bottom) B220 and CD43/S7 expression on IgM-negative lymphocytes, as gated in the upper panels. Numbers indicate the percentage of total lymphocytes that belong to the B220+CD43−sIgM− pre-B cell population. Immunity 1997 7, 765-775DOI: (10.1016/S1074-7613(00)80395-7)

Figure 3 Southern Blot Analysis of κ-Expressing Splenocytes from Igi Mice (A) The 3-83κi and wild-type Igκ alleles. Probe Eco-Eco hybridizes to 5.6 kb and 5.0 kb SacI bands of the targeted and wild-type alleles, respectively. Secondary Vκ-Jκ rearrangements (curved arrows) cause deletion or inversion of the intervening region, changing the size of the SacI hybridizing fragments and resulting in decreased intensity of the specific bands upon hybridization. (B) Example of hybridization of probe Eco-Eco to SacI digested DNA from either tail or κ+ splenocytes of 3-83Hi;3-83κi and 3-83κi mice. The 3-83κi and germline (gl) hybridizing bands are indicated. Lanes: (1) κ+ from 3-83Hi;3-83κi, (2) tail from 3-83Hi;3-83κi, (3) κ+ from 3-83κi, (4) tail from 3-83κi. (C) The same blot of (B) hybridized with an IL-4 gene-specific probe. This hybridization was utilized for the relative quantitation of the 3-83κi and germline bands in κ+ B cells compared to tail DNA samples. Immunity 1997 7, 765-775DOI: (10.1016/S1074-7613(00)80395-7)

Figure 4 PCR Analysis of RS Recombination in Splenocytes from Igi Mice (A) Scheme of the 3-83κiJκ− and 3-83κi loci and the strategy employed for the PCR analysis of RS recombination. RSS of Vκ regions, IRS1, and RS sequences are depicted by open triangles. IRS2 sequence (not indicated) is 400 bp 3′ of IRS1. Black arrows, primers utilized in the analysis. S, SacI. (B) Electrophoretic analysis of the PCR reactions performed with primers a and c. Template DNA was prepared from either tail or κ-expressing splenocytes isolated by cell sorting or from a total spleen cell population containing 10% λ+ cells. DNA from 5 × 104 κ+ cells was utilized per reaction. Tail DNA and total spleen DNA were, respectively, approximately 0.5 μg per reaction. Lanes: (1) 1 kb ladder DNA marker, (2) wild-type tail DNA, (3) κ+ splenocyte DNA from wild-type mice, (4) spleen (10% λ+ cells) DNA from 3-83Hi;3-83κiJκ− mice, (5) κ+ splenocyte DNA from 3-83Hi;3-83κiJκ− mice, (6) 1 kb ladder DNA marker. Arrow, the expected 1.04 kb band. (C) Electrophoretic analysis of PCR reactions performed with primers b and c. Template DNA was as in (B). Lanes are: (1) 1 kb ladder; (2–5), respectively, 41 ng, 2.5 ng, 0.6 ng , 0.16 ng of DNA from κ+ splenocytes of 3-83Hi;3-83κi mice; (6) 1 kb ladder; (7–10), respectively, 160 ng, 10 ng, 2.5 ng, 0.6 ng of DNA from κ+ splenocytes of B1-8Hi;3-83κi mice; (11) 1 kb ladder; (12) 1 mg of tail DNA from 3-83Hi;3-83κi mice; (13) spleen (10% λ+ cells) DNA from 3-83Hi;3-83κi mice. Arrows, the expected 180 bp band. Immunity 1997 7, 765-775DOI: (10.1016/S1074-7613(00)80395-7)

Figure 6 Intracellular κ Chain Expression in B220+CD43−sIgM− Cells from 3-83Hi;3-83κi Mice The B220+CD43−sIgM− cell population was sorted with a FACstar flow cytometer from bone marrow of 3-83Hi;3-83κi mice stained as in Figure 5 and then stained for intracellular κ chains. Flow cytometric analysis of intracellular κ chain expression versus forward size scatter is shown. The numbers indicate the percentage of the large κ+, small κ+, and small κ− cell fractions in the population analyzed. Immunity 1997 7, 765-775DOI: (10.1016/S1074-7613(00)80395-7)