Creation of mice expressing human antibody light chains by introduction of a yeast artificial chromosome containing the core region of the human immunoglobulin kappa locus.

We have previously described a strategy for integrating selectable marker genes into yeast artificial chromosomes (YACs) to facilitate their transfer into embryonic stem (ES) cells. Here we apply this technology to create mice carrying the core region of the human immunoglobulin (Ig) kappa light chain locus. A YAC was isolated which contains a 300 kb insert spanning three V kappa segments, the J kappa cluster, the C kappa region and extending downstream of the Kde element. After modification of this YAC to integrate the selectable neo marker gene, the YAC was introduced into ES cells by protoplast fusion. Several ES cell clones were obtained which appeared to harbor one complete copy of the YAC while retaining little or no other yeast DNA. The ES cells were injected into blastocysts and the chimaeric mice were shown to rearrange the introduced human light chain genes with the resultant production of antibodies containing human kappa light chains in the serum.

Targeted alterations in yeast artificial chromosomes for inter-species gene transfer.

In order to facilitate alterations of large DNA molecules for their introduction into mammalian cells we have characterised the mechanism of site-specific modifications in yeast artificial chromosomes (YACs). Newly developed yeast integration vectors with dominant selectable marker genes allow targeted integration into left (centromeric) and right (non-centromeric) YAC arms as well as alterations to the human derived insert DNA. In transformation experiments, integration proceeds exclusively by homologous recombination although yeast prefers linear ends of homology for predefined insertions. Targeted regions can be rescued which expedite the cloning of internal human sequences and the identification of 5' and 3' YAC/insert borders. Integration of the neomycin resistance gene into various parts of the YAC allowed the transfer and stable integration of large DNA molecules into a variety of mammalian cells including embryonic stem cells.

Subtle differences in antibody responses and hypermutation of lambda light chains in mice with a disrupted chi constant region.

Analysis of lambda light chain use in normal mice is made difficult by the dominant chi light chain repertoire. We produced mice rendered deficient in chi light chain expression by gene targeting and focused on questions concerned with the generation of lambda light chain diversity. Whilst these mice compensate the chi deficiency with increased lambda liters, and their Ig level is therefore not significantly reduced, they show major differences in immunization titers, germinal center (GC) development and somatic hypermutation. After immunization, using antigens that elicit a restricted IgL response in normal mice, we obtained in the chi-/- mice elevated primary antibody titers but a subsequent lack in titer increase after repeated antigen challenge. Analysis of the Peyer's patches (PP) revealed a dramatically reduced cell content with rather small but highly active GC. Flow cytometric analysis showed different cell populations in the PP with enriched peanut agglutinin (PNA)hi/CD45R(B220)+ B cells, implying that the apparent compensation for the lack of lambda light chain expression involves the GC microenvironment in cell selection, the initiation of hypermutation and high affinity expansion. The three V lambda genes, V1, V2 and Vx, are mutated in the GC B cells, but show no junctional diversity. In contrast, a reduced rate of V lambda hypermutation is found in the hybridoma antibodies, which appears to reflect a selection bias rather than structural constraints. However, mechanisms of somatic mutation and specificity selection can operate with equal efficiency on the few V lambda genes.

Exit of the pre-TCR from the ER/cis-Golgi is necessary for signaling differentiation, proliferation, and allelic exclusion in immature thymocytes.

A major issue is whether surface expression of the pre-TCR is necessary for signaling the development of immature thymocytes. To address this question, we generated transgenic mice expressing a TCRbeta chain that had a strong endoplasmic reticulum (ER) retrieval signal (TCRbetaER) and that was expressed intracellularly but failed to reach the cell surface. In TCRbetaER transgenic mice, there was a failure of allelic exclusion. Also, the transgene failed to rescue the developmental defects observed in TCRbeta-null mice. In contrast, TCRbeta transgenes with a mutant ER retrieval sequence or lacking this sequence signaled efficient allelic exclusion and suppressed the TCRbeta-/- defect. These data show that exit of the pre-TCR from the ER/cis-Golgi is required for progression through the double-negative thymocyte checkpoint.

Raf regulates positive selection.

T cell development is regulated by extracellular signals that mediate cellular proliferation and differentiation via specific signal transduction pathways. To determine the importance of the mitogen-activated protein kinase (MAP kinase) pathway in thymocyte development, we analyzed transgenic mice expressing dominant negative Raf (DN Raf) and a constitutively active v-Raf under the control of the p56lck proximal promoter. DN Raf had a profound effect on T cell receptor (TCR)-mediated signaling events as assessed by the inhibition of mitogen-induced proliferation of thymocytes in vitro. Overall thymocyte numbers were decreased by at most twofold from nontransgenic littermates. Positive selection was inhibited in DN Raf transgenic mice, as evidenced by both reduced numbers of mature thymocytes and a decrease in CD8+ thymocytes in female mice doubly transgenic for DN-Raf and a class I-restricted H-Y TCR. In contrast, the differentiation of double-positive thymocytes to single-positive thymocytes was enhanced in H-YTCR transgenic mice expressing constitutively active Raf (v-Raf). Thus, Raf regulates positive selection in the thymus.

Truncation of the Mll gene in exon 5 by gene targeting leads to early preimplantation lethality of homozygous embryos.

The mixed lineage leukemia gene (MLL) was originally identified through its involvement in reciprocal translocations in leukemias. MLL codes for a large multidomain protein and bears homology to the Drosophila developmental control gene trithorax in two small domains in the amino terminal region, the central zinc finger domain and the carboxy SET domain. Like the Drosophila trx, MLL has also been shown to be a positive regulator of Hox gene expression. We have targeted Mll (the murine homologue of MLL) in exon 5 causing expression of three truncated in-frame Mll transcripts. These transcripts retain all or some of the AT hook motifs and the DMT domain. This mutant allele causes early in vivo preimplantation lethality of homozygous embryos prior to the 2-cell stage. Embryos cultured in vitro progress to the 2-cell stage, but further development is arrested. The heterozygotes exhibit mild skeletal defects as well as defects in some neuroectodermal derivatives.

Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development.

We have generated Cbfa1-deficient mice. Homozygous mutants die of respiratory failure shortly after birth. Analysis of their skeletons revealed an absence of osteoblasts and bone. Heterozygous mice showed specific skeletal abnormalities that are characteristic of the human heritable skeletal disorder, cleidocranial dysplasia (CCD). These defects are also observed in a mouse Ccd mutant for this disease. The Cbfa1 gene was shown to be deleted in the Ccd mutation. Analysis of embryonic Cbfa1 expression using a lacZ reporter gene revealed strong expression at sites of bone formation prior to the earliest stages of ossification. Thus, the Cbfa1 gene is essential for osteoblast differentiation and bone formation, and the Cbfa1 heterozygous mouse is a paradigm for a human skeletal disorder.