MOLECULAR REGULATION OF COMPROMISED B LYMPHOPOEISIS IN AGED MICE (original) (raw)
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British Journal of Haematology, 1990
A method has been developed to use the polymerase chain reaction to amplify and sequence the chain determining region 3 (CDR 3) of the human immunoglobulin heavy-chain gene, and to use the sequence as a marker for rare neoplastic B lymphocytes. Consensus primers for the Variable and Joining regions of the gene were constructed and shown to enable efficient amplification, directed cloning, and sequencing of CDR 3. Using leukaemic cell line PFMC as a test system, CDR 3 was sequenced, specific primers synthesized, and PFMC DNA was detected down to a dilution of 1:1300 in DNA from normal lymphocytes. This strategy should be useful for monitoring therapy and detecting early disease relapse in B lymphoproliferative disease.
Neoteny in Lymphocytes: Rag1 and Rag2 Expression in Germinal Center B Cells
Science, 1996
These PCR cycles were confirmed to be in the exponential phase of the amplification. PCR products were electrophoresed on 7.5% polyacrylamide gel and visualized by Southern (DNA) blotting using 32 P-labeled probes, the Dde I-Dde I 163-base pair (bp) internal fragment of RAG-1 cDNA, the Pst I-HinfI 124-bp internal fragment of RAG-2 cDNA, and the entire coding region of GAPDH cDNA. Hybridized filters were exposed to Fuji imaging plate (Fuji Film) for 2 days and visualized by a Bioimaging Analyzer, BAS 1000 (Fuji Film). RAG-1 and RAG-2 cDNAs were given by D. G. Schatz (Yale University, New Haven, CT). 19. Mouse B cells were prepared by treating spleen cells from male C3H/HeN mice (8 to 10 weeks of age, Japan Charles River) with 1/1000-diluted mAb to Thy 1.2 mAb (SeroTec), followed by incubation with low-toxic rabbit complement (Cederlane) as described [K. Haruna et al., Cell. Immunol. 151, 52 (1993)]. The B cells (3 ϫ 10 6 cells per milliliter) were cultured with LPS (20 g/ml) from Escherichia coli 055 B5 (Sigma) and mouse recombinant IL-4 (500 U/ml; PeproTech) in 1 ml of RPMI-1640 medium containing 10% fetal bovine serum, 10 M 2-mercaptoethanol, penicillin G (100 U/ml), and streptomycin (50 g/ml). In some cases, mAb to mouse CD40 (1 g/ml; rat mAb LB429 presented by N. Sakaguchi, Kumamoto University, Japan) or F(abЈ) 2 fragment of goat antibody to mouse heavy chain (10 g/ml; Cosmo Bio) plus 1 mM 8-mercaptoguanosine (Sigma) was used as a stimulus. Because it was confirmed that RAG expression peaked on day 2 of the culture and declined thereafter, all cultures were carried out for 2 days. In in vivo experiments, mice were immunized with 20 g of TNP-KLH and 0.45 mg of alum in each hind footpad. Inguinal or popliteal LN cells from three mice were pooled on day 0, 6, and 8 postimmunization and assessed for the expression of RAG-1 and RAG-2 mRNA (18). 20. Culture plates of 100 mm diameter were coated with 100 g/ml of mAb to mouse IgD (Biosys). Sixty million mouse spleen cells that had been depleted of T cells and erythrocytes were placed in the plate and incubated for 1 hour at room temperature. Then the plate was gently washed with phosphate-buffered saline four times to remove nonadherent cells. It was confirmed by flow cytometric analysis (FACScan) that adherent cells recovered from the plate were more than 99% positive for sIgD and B220. 21. Monoclonal antibodies to mouse RAG-1 (G109-256.2, mouse IgG2b) and RAG-2 (G110-461, mouse IgG2b) were obtained from Pharmingen. A myeloma-derived murine IgG2b (MOPC 195) was used as an irrelevant negative control (ICN Biomedicals). These mAbs were biotinylated under the same conditions, using a biotinylation kit (American Qualex). Staining of RAG proteins in thymocytes or cultured B cells was carried out as described (10), with some modifications. Briefly, the cultured cells were fixed on glass slides in methanol-acetone (1:1) for 5 min, rehydrated in phosphate-buffered saline, and preblocked for 1 hour with TBST [10 mM tris (pH 8.0), 150 mM NaCl, and 0.05% Tween 20] containing 1% bovine serum albumin (BSA) and MOPC 195 (50 g/ml). The slides were then incubated in TBST containing 1% BSA and one of each biotinylated mAb (5 g/ml) for 1 hour at room temperature. Slides were then washed three times in TBST and reacted for 1 hour with rhodaminated avidin (2 g/ml; Sigma) in TBST containing 1% BSA. For the immunofluorescent staining of LN sections, 6-m-thick cryosections mounted on slides were allowed to air dry for 15 min and were fixed in ice-cold acetone for 10 min. After rehydration and preblocking as described above, the sections were treated with biotinylated anti-RAG-1 (5 g/ml) for 1 hour, followed by double-staining with rhodaminated avidin (2 g/ml) and FITC-PNA (4 g/ml; Seikagaku Kogyo) for 40 min. All reagents were diluted in TBST containing 1% BSA. After washing with TBST, samples were finally mounted with low-fluorescent glycerol and cover slip protection, and were observed with a Zeis fluorescence microscope. 22. This work was supported in part by a grant-in-aid from the Ministry of Education, Science and Culture of Japan. We thank H. Kagawa for excellent secretarial assistance.
European journal of …, 1994
To clone the rat CD5 gene we first produced two rat CD5 probes. The probes were obtained by polymerase chain reaction (PCR) on rat genomic DNA using primers designed on conserved regions between mouse and human CD5. The screening of a rat cDNA library at high stringency using these probes resulted in a 1.5-kb positive clone. The DNA sequence of this clone confirmed its CD5 nature, but the clone appeared to lack part of the 5′ and part of the 3′ end. These missing 5′ and 3′ ends were obtained by PCR on rat thymus RNA. By ligating these PCR products to the original 1.5-kb CDM8 clone, a full-length rat CD5 gene was constructed. The full-length clone showed high identity with mouse and human CD5; however, at the 5′ site of the gene a region of 36 nucleotides is present which is not seen in either mouse or human CD5. We have evidence that this sequence is a normal constituent of the rat CD5 gene: first, it is in frame with the rest of the CD5 coding sequence; second, it does not contain a stop codon; and third, it is also present in the CD5 gene of other rat strains. We transfected the full-length CD5 construct in COS cells and demonstrated that indeed the CD5 protein is recognized by MRC OX19. Although we showed that CD5 mRNA is present in rat B cells, extensive flow cytometry analysis using MRC OX19 as antibody failed to detect B cells expressing significant levels of CD5 on their cell surface compared to other B cells in any tissue or cell suspension tested from a variety of rat strains. This is in contrast with the mouse where a distinct population of B cells (B-la cells) can be found expressing more CD5 than the other B cells. Either B-1 cells are not present in rats or CD5 is not the right phenotypic marker for rat B-1 cells. It still remains to be investigated whether a population of B cells with functions similar to those of murine B-1 cells is present in rats.
Journal of Immunological Methods, 2003
We established a new tool to perform semiquantitative and qualitative screening for VH gene usage frequency during IgH rearrangements in human B-lymphocytes. In two separate multiplex PCRs, the rearranged VDJ regions were amplified with VH family-specific primers labeled with different fluorescent dyes (FAM, HEX, NED, or ROX). The relative amount of each of the particular VH family products and their ratios were determined by fragment analysis on a ABI PRISM 377 sequencer. We verified that the fluorescent multiplex PCR (FMPCR) shows high specificity and sensitivity, acceptable reproducibility and reliability. Data obtained were well in agreement with results revealed by sequencing following single-cell PCR. Ten healthy volunteers showed a comparable semiquantitative VH family distribution. The FMPCR also correctly detected a monoclonal peak in a CLL patient. Thus, labeling primers with various fluorescent dyes allows for an assessment of VH family usage and an immediate determination of the involved VH gene family if any clonal peaks are present. This method provides a quick, easy, and reliable tool for VH repertoire screening of larger populations of patients suffering from diseases with changes in the VH repertoire allowing for selection of cases worth a more detailed and cumbersome sequence analysis later on.