Pi-Chen Yam

Functional Analysis of Iα Promoter Regions of Multiple IgA Heavy Chain Genes

The 13 nonallelic IgA H chain genes of rabbit are differentially expressed in vivo. They can be grouped into those expressed at high levels (Cα4, Cα5, Cα6, Cα9, Cα10, Cα12, and Cα13), those expressed at low levels (Cα1, Cα2, Cα7, and Cα11), and those that are not expressed (Cα3 and Cα8). We tested whether the differential in vivo expression is due to differential responses of the Iα promoters to TGF-β stimulation. We stimulated the rabbit B cell line 55D1 with TGF-β and, using single-cell RT-PCR, found that expression of germline (GL) transcripts of α3 and α8 could not be induced. By luciferase reporter gene assay and EMSA we found that the promoters of the unexpressed isotypes Cα3 and Cα8 are defective, thereby explaining the absence of IgA3 and IgA8 in vivo. When comparing the promoter activities of the other isotypes we found that the activities did not reflect the degree of in vivo expression. Instead, the promoters of the isotypes expressed at high or low levels promoted expression of the luciferase gene to a similar degree, except for the Iα4 promoter, which had much higher activity. Also the degree to which TGF-β induced GL expression of the various isotypes in 55D1 B cells did not reflect in vivo expression. However, most of the TGF-β-stimulated cells expressed GL mRNA of multiple isotypes; no isotype was expressed preferentially. These results suggest that the final switch to a single isotype is regulated in a step subsequent to GL transcription, rather than by induction of GL transcripts by the Iα promoter.

B lymphocyte deficiency in IgH‐transgenic rabbits

We developed IgH‐transgenic rabbits carrying a productive VDJ‐Cμ Tg and found the rabbits were B cell‐deficient, with a 50–100% reduction in serum IgM and IgG levels. The bone marrow of newborn Tg rabbits contained severely reduced levels of preB cells and almost no B cells. The few preB cells present in the bone marrow were large, cycling cells that expressed the VDJ‐Cμ Tg, indicating that the block in B cell development likely occurred at or before the transition from large (early) preB to small (late) preB cells. By immunoprecipitation, the Tg μ‐chain paired with VpreB and λ5, suggesting that the B cell deficiency is not due to an inability to form a preB cell receptor. Despite the block in B cell development, a few B cells, expressing predominantly endogenous μ‐chains, began the second stage of development in GALT. B cells were localized in and beneath the follicle‐associated epithelium of GALT prior to B cell follicle formation, suggesting to us that B cell follicle formation is initiated near the follicle‐associated epithelium, possibly through contact with intestinal microbiota. These IgH‐Tg rabbits should provide a useful model for studies of B cell development both in bone marrow and in GALT.

RABBIT MAJOR HISTOCOMPATIBILITY COMPLEX IV. EXPRESSION OF MAJOR HISTOCOMPATIBILITY COMPLEX CLASS II GENES

The rabbit MHC class II DP, DQ, and DR alpha and beta chain genes were transfected into murine B lymphoma cells. The transfected cells expressed R‐DQ and R‐DR molecules on the cell surface but they did not express the R‐DP genes either on the cell surface or at the level of mRNA. Northern blot analyses showed that the R‐DP genes were expressed, albeit at low levels, in rabbit spleen. Similar analyses showed that the R‐DQ and R‐DR genes were expressed at high levels in rabbit spleen. A new monoclonal anti‐rabbit class II antibody, RDR34, has been developed and shown to react with the R‐DR transfected cells and not with the R‐DQ transfected cells. The previously described monoclonal anti‐rabbit class II antibody, 2C4, reacted with the R‐DQ transfected cells and not with the R‐DR transfected cells. Thus, 2C4 and RDR34 MAbs are specific for the R‐DQ and R‐DR molecules, respectively. Each of the antibodies reacted with approximately 50% of rabbit spleen cells as shown by immunofluorescent antibody studies.

Generation of rabbit monoclonal antibodies.

Rabbit hybridomas are gaining wide acceptance as serologic reagents to identify a variety of antigens, including proteins, small peptides, phosphorylated proteins, and polysaccharides. Rabbits make high-affinity IgG antibodies, all of which bind with high affinity to Protein A from Staphylococcus aureus and Protein G from Group G Streptococcus. Consequently, rabbit monoclonal antibodies of desired specificity can be rapidly detected using Protein A/G as secondary reagents. Here we describe the method for generating rabbit monoclonal antibodies using the rabbit hybridoma fusion partner 240E-1. The method begins with the immunization of rabbits and ends with the cloning the antigen-specific hybridomas.

Leukemia, Lymphoma, Embryonic Carcinoma and Hepatoma Induced in Transgenic Rabbits by the c-myc Oncogene Fused with Immunoglobulin Enhancers

Transgenic rabbits were developed with the rabbit c-myc proto oncogene fused to the immunoglobulin heavy or light chain enhancers, E µ or E κ .Rabbits transgenic for E µ -mycdeveloped leukemia very early in life, 17–21 days of age, and were terminally ill. The disease was diagnosed as acute lymphoblastic leukemia resembling childhood leukemia. The white blood cell counts were increased as much as 100-fold, the spleens were enlarged and various organs were infiltrated by the neoplastic lymphocytes. Analysis of the DNA showed that immunoglobulin heavy and light chain genes were rearranged, showing the leukemia to be of the B cell type. Since several different rearranged VDJ genes could be cloned from each of three rabbits we concluded that the leukemias were polyclonal. The blood lymphocytes, as well as the cells cloned from various tissues, appear to be lymphoblastic, that is, they are large, have a large nucleus and have very little cytoplasm. These cells do not express surface immunoglobulin, but they have a low concentration of cytoplasmic IgM and they secrete small amounts of light chain.