Hybridomas were produced from spleen cells of kappa transgenic mice to investigate expression of the transgenic kappa gene, its effect on allelic exclusion and its effect on the control of light-chain gene rearrangement and expression. Our results show that the transgene is expressed normally and that the production of a complete immunoglobulin molecule turns off light-chain gene rearrangement.

The hematopoietic stem cell (HSC) is defined as a cell that can either self-replicate or generate daughter cells that are destined to commit to mature cells of different specific lineages. Self-replication of the most primitive HSC produces daughter cells that possess a long (possibly unlimited) clonal lifespan, whereas differentiation of HSC produces daughter cells that demonstrate a progressive reduction of their clonal lifespan, a loss of multilineage potential, and lineage commitment. Previous studies indicated that the proliferation of HSC ex vivo favors differentiation at the expense of self-replication, eventually resulting in a complete loss of HSC. In contrast, transplantation studies have shown that a single HSC can repopulate the marrow of a lethally irradiated mouse, demonstrating that self-renewal of HSC occurs in vivo. Thrombopoietin (TPO) has been shown to function both as a proliferative and differentiative factor for megakaryocytes and as a survival and weakly proliferative factor for HSC. Our studies focused on the effects of exogenous TPO on HSC in mouse long-term bone marrow cultures (LTBMC). Previous results indicate that HSC decline in LTBMC in the absence of TPO. In contrast, the continuous presence of TPO resulted in the generation of both long-and short-term repopulating HSC as detected by an in vivo competitive repopulation assay. HSC were generated over a 4-month period at concentrations similar to normal bone marrow. Our results demonstrate that TPO can mediate the self-replication of HSC in LTBMC, and provide proof that HSC can self-replicate ex vivo.

Transgenic mice were produced by microinjection of a rearranged, functional immunoglobulin κ gene into fertilized mouse eggs and implantation of the microinjected embryos into foster mothers. Mice that integrated the injected gene were mated and the DNA, RNA and serum κ chains of their offspring were analysed. The data from offspring of three different transgenic mice indicate that the microinjected gene is expressed in the spleen, but not the liver of mice which inherited the injected gene.Immunoglobulin variable (V) and constant (C) region genes must be rearranged into close proximity before expression into a functional immunoglobulin protein can occur 1,2 . The functional rearrangement process takes place only in the B-lymphocyte lineage. To investigate the control of immuno-globulin gene expression we have produced transgenic mice by microinjection 3 of an immunoglobulin gene into fertilized mouse eggs. Microinjection of a gene into an embryo introduces that gene into every cell of the resultant mouse and often leads to efficient expression of the injected gene [4][5][6] . The presence of the injected gene in germ cells allows offspring of transgenic mice to inherit the injected gene. Compared with cell transfection experiments, this approach has the advantages of avoiding potential problems due to the transformed phenotype of cultured cells and allowing direct comparison of expression in different tissues that have the injected gene at the same location in the genome.In our experiments the injected gene is the functional κ gene of the myeloma . Its variable region, V κ M.21, is rearranged next to the joining gene segment J κ 2 (ref. 8) and is contained in the plasmid pB1-14 (Fig. 1). The V κ M.21 portion of the microinjected gene provides a means to distinguish the expression of the introduced gene from that of the endogenous κ genes, both on the RNA and protein levels. The V κ M.21 gene is a member of the V κ 15 family of V κ genes 9,10 . This gene family has approximately ten members which are sufficiently homologous to cross-hybridize using a V κ M.21 probe 11 . V κ genes of this family are probably rearranged and expressed in some B lymphocytes in normal mice, and transcripts from endogenous genes might be expected to confound the detection of transcripts from the injected gene. However, transcription of homologous endogenous V κ 15 were expressed in a tissue at a normal rate in most cells or at a high rate in a subpopulation of cells. The MOPC-21 κ protein is also sufficiently distinct in molecular weight 12 and isoelectric point (see below) that it can be distinguished from other κ chains in mouse serum, provided it is produced and secreted in some quantity. Analysis of transgenic mice containing the pB1-14 gene shows that expression of the microinjected κ gene is apparent on both the RNA and protein levels. HHS Public Access Production of transgenic miceDNA for injection was prepared as described in Fig. 1. Eggs from (C57BL/6 × SJL)F 1 mice were fertilized with F 1 sperm. The male pronuclei o...