Marker protein expression in dκGT-hCD4 and Cκ-iYFP knock-in mice. (A) Schematic of the dκGT-hCD4 and Cκ-iYFP knock-in mutations at the Igκ locus. Both the proximal and distal germline transcript promoters are shown as arrows. Previously defined splicing patterns of both transcripts are depicted as dashed lines. The hCD4 cDNA is followed by an SV40 intron and polyA sequence. Black triangles represent the positions of loxP sites remaining in the locus after Cre recombinase-mediated deletion of a neomycin resistance gene. (B) Flow cytometric analysis of hCD4 or YFP marker expression in developing bone marrow B cells from heterozygous dκGT-hCD4 or Cκ-iYFP knock-in mice. Harvested bone marrow was labeled with antibodies to delineate B cell developmental subsets and mark hCD4-expressing cells (in dκGT-hCD4 mice only). CLP through fraction F are shown. The percentage of marker-positive cells at each developmental stage is shown. Control C57/BL6 mice had no detectable expression of either marker (data not shown). Data are representative of at least five independent experiments analyzing two to four mice per experiment. (C) Flow cytometric analysis of hCD4 or YFP marker expression in splenic B cells from heterozygous dκGT-hCD4 or Cκ-iYFP knock-in mice. Harvested splenocytes were labeled with antibodies to delineate B cell developmental subsets and mark hCD4-expressing cells (in dκGT-hCD4 mice only). Transitional 1 (T1), transitional 2 (T2), transitional 3 (T3), follicular (Fo), marginal zone (MZ), and B-1 B cell subsets are shown along with the percentage of marker positive cells at each developmental stage. Data are representative of at least two independent experiments analyzing two to four mice per experiment.

The distal germline Jκ promoter is far more active than the proximal germline Jκ promoter in developing pre-B cells. (A) Schematic of the Jκ cluster. The transcription start sites for both the distal and proximal promoters are shown as arrows above the diagram along with the deduced splicing pattern of the germline transcripts in dashed lines. The unfilled box at the left represents the first exon of one of the distal-promoter transcripts (D1), and the gray box the first exon of the other (D2). The two different splice donor sites are indicated. Approximate primer locations for both real-time RT-PCR (B) and gel-based RT-PCR (C) of Jκ transcripts are shown as small arrows. (B) Quantitative real-time PCR analysis of spliced Jκ germline transcripts in sorted B cell subsets from wild-type mice. Distal promoter transcripts were quantified using primer #3 (transcript D1) or primer #9 (transcript D2) paired with the reverse primer in Cκ. Proximal promoter derived transcripts (transcript P) were quantified using the same primer pair as for the alternatively spliced distal transcript D2. Harvested bone marrow from C57/BL6 mice was labeled as in (Fig. 1B) and sorted by flow cytometry into the indicated fractions before harvesting cells for RNA isolation. Cntl represents a positive control RNA sample from AMuLV transformed pro-B cells. Values were normalized to Actin transcript abundance (± SD). Data are representative of two independent experiments. (C) RT-PCR analysis of Jκ germline transcripts in primary pre-B cells. Pre-B cells from wild-type mice were sorted and processed for RNA isolation and subsequent RT-PCR. Various forward primers (numbered arrows) were paired with a common reverse primer in the Cκ exon. An ethidium bromide-stained agarose gel analysis of the resultant products is shown. Lane marked L is a standard DNA size ladder. Numbers above the lanes indicate the forward primer used according to the diagram in (A). Data are representative of two independent experiments. (D) 5′RLM-RACE analysis of germline Igκ transcripts in developing primary B cells. Primary cells were harvested and sorted from wild-type C57/BL6 mice and processed for RNA isolation and subsequent 5′RLM-RACE (strategy shown in Fig. S3). The graph represents the ratio of transcripts initiating from the distal and the proximal Igκ germline transcript promoters. No significant quantities of either transcript were detected in thymocytes or water controls. Data are representative of two independent experiments, with each PCR done in triplicate.

Germline transcription at the Jκ cluster is biallelic. Flow cytometric analysis of marker expression in developing B cells from dκGT-hCD4/Cκ-iYFP compound heterozygous mice. Harvested bone marrow was labeled with antibodies to delineate developing B cell subsets and mark hCD4 expression. The progression of cells from the pro-B to the small resting pre-B cell stages is shown. Small pro-B cells were defined as forward scatter low CD43⁺B220⁺ while small pre-B cells were forward scatter low CD43⁻B220⁺. The intermediate stages represent forward scatter high cells (cycling) with intermediate levels of CD43 progressing from high to low. Data are representative of two independent experiments analyzing three mice each.