Knock-in of nuclear localised beta-galactosidase reveals that the tyrosine phosphatase Ptprv is specifically expressed in cells of the bone collar.

Ptprv is a member of the transmembrane tyrosine phosphatase gene family reported to be expressed in osteoblasts and gonads. To better define the developmental and tissue specificity of Ptprv expression, we generated knock-in mice expressing a nuclear localised beta-galactosidase reporter under the control of resident Ptprv regulatory elements. Histochemical staining of Ptprv-nLacZ mice revealed that Ptprv expression is readily detectable in the foetal gonadal ridge of both sexes and in adult gonads where it is localised to Sertoli cells of the testis and celomic epithelial cells of the ovaries. During early limb development, Ptprv expression is prominent in the apical ectodermal ridge of the limb bud. At latter stages of development, Ptprv is predominantly expressed in the perichondrial and periosteal region of long bones, known as the bone collar. In contrast to previous indications from in vitro studies, there is little if any expression in mature osteoblasts in vivo. Analysis of Ptprv mRNA localisation by in situ hybridization in parallel with molecular markers of chondrocytes and osteoblasts confirmed the specific expression of Ptprv in immature bone collar cells. The specificity of Ptprv expression in these cells may be a useful tool to elucidate their role in the transition of skeletal elements from cartilage template to bone.

Artificial chromosomes: ideal vectors?

Artificial chromosomes are DNA molecules of predictable structure, which are assembled in vitro from defined constituents that behave with the properties of natural chromosomes. Artificial chromosomes were first assembled in budding yeast and have since been useful in many aspects of yeast genetics. Several attempts have been made at building artificial chromosomes in mammals, although these have been met with limited success. Consequently, mini-chromosomes of defined structure have been developed to address questions regarding mammalian chromosome function and for biotechnological applications. Here we review progress in these areas and consider how it influences plans to build artificial chromosomes in plants and parasites.

A structurally defined mini-chromosome vector for the mouse germ line.

Yeast artificial mini-chromosomes have helped to define the features of chromosome architecture important for accurate segregation and replication and have been used to identify genes important for chromosome stability and as large-fragment cloning vectors. Artificial chromosomes have been developed in human cells but they do not have defined, experimentally predictable structures. Fragments of human chromosomes have also been introduced into mice and in one case passed through the germ line. In these experiments, however, the structure and sequence organization of the fragments was not defined. Structurally defined mammalian mini-chromosome vectors should allow large tracts of DNA to be introduced into the vertebrate germ line for biotechnological purposes and for investigations of features of chromosome structure that influence gene expression. Here, we have determined the structure and sequence organization of an engineered mammalian mini-chromosome, ST1, and shown that it is stably maintained in vertebrate somatic cells and that it can be transmitted through the mouse germ line.

Greatly reduced efficiency of both positive and negative selection of thymocytes in CD45 tyrosine phosphatase-deficient mice.

The T cell repertoire is shaped by positive and negative selection of thymocytes. TCR-mediated signals that determine these selection processes are only partly understood. The CD45 tyrosine phosphatase has been shown to be important for signal transduction through the TCR, but there has been disagreement about whether CD45 is a positive or negative regulator of TCR signaling. Using CD45-deficient mice expressing transgenic TCR, we show that in the absence of CD45 there is a large increase in the thresholds of TCR stimulation required for both positive and negative selection. Our results conclusively demonstrate that in double-positive thymocytes CD45 is a positive regulator of the TCR signals that drive thymic selection events.

Transchromosomal mouse embryonic stem cell lines and chimeric mice that contain freely segregating segments of human chromosome 21.

At least 8% of all human conceptions have major chromosome abnormalities and the frequency of chromosomal syndromes in newborns is >0.5%. Despite these disorders making a large contribution to human morbidity and mortality, we have little understanding of their aetiology and little molecular data on the importance of gene dosage to mammalian cells. Trisomy 21, which results in Down syndrome (DS), is the most frequent aneuploidy in humans (1 in 600 live births, up to 1 in 150 pregnancies world-wide) and is the most common known genetic cause of mental retardation. To investigate the molecular genetics of DS, we report here the creation of mice that carry different human chromosome 21 (Hsa21) fragments as a freely segregating extra chromosome. To produce these 'transchromosomal' animals, we placed a selectable marker into Hsa21 and transferred the chromosome from a human somatic cell line into mouse embryonic stem (ES) cells using irradiation microcell-mediated chromosome transfer (XMMCT). 'Transchromosomal' ES cells containing different Hsa21 regions ranging in size from approximately 50 to approximately 0.2 Mb have been used to create chimeric mice. These mice maintain Hsa21 sequences and express Hsa21 genes in multiple tissues. This novel use of the XMMCT protocol is applicable to investigations requiring the transfer of large chromosomal regions into ES or other cells and, in particular, the modelling of DS and other human aneuploidy syndromes.

The Rho-family GTP exchange factor Vav is a critical transducer of T cell receptor signals to the calcium, ERK, and NF-kappaB pathways.

Vav is a GTP/GDP exchange factor (GEF) for members of the Rho-family of GTPases that is rapidly tyrosine-phosphorylated after engagement of the T cell receptor (TCR), suggesting that it may transduce signals from the receptor. T cells from mice made Vav-deficient by gene targeting (Vav-/-) fail to proliferate in response to TCR stimulation because they fail to secrete IL-2. We now show that this is due at least in part to the failure to initiate IL-2 gene transcription. Furthermore, we analyze TCR-proximal signaling pathways in Vav-/- T cells and show that despite normal activation of the Lck and ZAP-70 tyrosine kinases, the mutant cells have specific defects in TCR-induced intracellular calcium fluxes, in the activation of extracellular signal-regulated mitogen-activated protein kinases and in the activation of the NF-kappaB transcription factor. Finally, we show that the greatly reduced TCR-induced calcium flux of Vav-deficient T cells is an important cause of their proliferative defect, because restoration of the calcium flux with a calcium ionophore reverses the phenotype.

Molecular requirements for lineage commitment in the thymus--antibody-mediated receptor engagements reveal a central role for lck in lineage decisions.

Recent experiments in our laboratory have focused on the receptor engagements required for the differentiation of fully mature, single positive thymocytes from their double positive precursors. We have used a novel approach which involves the ligation of surface receptors on immature thymocytes with genetically engineered F(ab1)2 reagents, which, unlike conventional antibodies, do not aggregate the CD3 complex to such an extent as to induce extensive deletion of these cells. The experimental data presented in this review indicate that differentiation of the two mature CD4 and CD8 lineages occurs in response to distinct intracellular signals induced by particular receptor engagements. The data suggest that the tyrosine kinase p56lck (lck) plays a crucial role in determining lineage choice, in that maturation of thymocytes into the CD4 lineage occurs upon recruitment of active lck to the T-cell receptor (TCR)/CD3 complex, whereas CD8 maturation can be induced by CD3 ligation in the absence of co-receptor-mediated lck recruitment. A central role for lck activity in determining the threshold for differentiation of the CD4 lineage is revealed in experiments with thymi deficient for a regulator of lck activity, CD45. A model of thymocyte differentiation is presented in which we propose that the relative balance of signals delivered by TCR engagement and lck activation determines lineage choice.

Hierarchical interactions of control elements determine CD8alpha gene expression in subsets of thymocytes and peripheral T cells.

CD4 and CD8 are crucial for the development and function of T cells. An intergenic deoxyribonuclease I hypersensitive site region (cluster CIII) directs expression in mature CD8 T cells only. Here, we show that two further independent regions from the CD8 gene locus in conjunction with cluster CIII restore transgene expression in appropriate immature thymocytes. Deletion of two of the intergenic cluster CIII DNaseI-HSS in homozygous mutant mice affects expression of CD8alphaalpha homodimers on intraepithelial T cells (IEL), particularly on the gammadeltaTCR+ subset. Surprisingly, none of the thymocyte or peripheral alphabetaTCR T cell subsets are affected by this mutation, indicating hierarchical activation of these elements within the different T cell subsets.

Tumorigenesis and a DNA repair defect in mice with a truncating Brca2 mutation.

Germline mutation of the BRCA2 gene carries a high risk of developing breast cancer. To study the function of this gene, we generated a mutation in Brca2 in mice. Unlike other mutations in the Brca2 gene, which are lethal early in embryogenesis when homozygous, some of our homozygous mutant mice survive to adulthood. These animals have a wide range of defects, including small size, improper differentiation of tissues, absence of germ cells and the development of lethal thymic lymphomas. Fibroblasts cultured from BrcaZ-/-embryos have a defect in proliferation that may be mediated by over-expression of p53 and p21Waf1/CIP1. We show that Brca2 is required for efficient DNA repair, and our results suggest that loss of the p53 checkpoint may be essential for tumour progression triggered by mutations in BRCA2.

A requirement for the Rho-family GTP exchange factor Vav in positive and negative selection of thymocytes.

The T cell repertoire is shaped by positive and negative selection of thymocytes that express low levels of T cell receptor (TCR) and both CD4 and CD8. TCR-mediated signals that determine these selection processes are only partly understood. Vav, a GDP-GTP exchange factor for Rho-family proteins, is tyrosine phosphorylated following TCR stimulation, suggesting that it may transduce TCR signals. We now demonstrate that mice lacking Vav are viable and display a profound defect in the positive selection of both class I- and class II-restricted T cells. In contrast, Vav is not essential for negative selection, though in its absence negative selection is much less effective. Vav may influence the efficiency of TCR-induced selection events by regulating the intracellular calcium flux of thymocytes.

Hematopoiesis in mice lacking the entire granulocyte-macrophage colony-stimulating factor/interleukin-3/interleukin-5 functions.

Interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-5 are major hematopoietic cytokines produced by activated T cells and exhibit similar biologic activities by signaling through a common receptor subunit (beta c). Mice lacking beta c show a pulmonary alveolar proteinosis-like disease and reduced numbers of peripheral eosinophils, which are explained by the lack of GM-CSF and IL-5 function, respectively. However, beta c-deficient hematopoietic cells do respond to IL-3 normally, probably through an additional beta subunit of the IL-3 receptor (beta IL3) that is present in the mouse. Thus, almost normal hematopoiesis in beta c-deficient mice may be caused by functional redundancy between IL-3 and GM-CSF. To clarify the role of the entire IL-3/GM-CSF/IL-5 system in hematopoiesis in vivo, we crossed the beta c mutant mice with mice deficient for IL-3 ligand to generate mice lacking the entire IL-3/GM-CSF/IL-5 functions. The double-mutant mice were apparently normal and fertile. The severity of the lung pathology in the beta c/IL-3 double-mutant mice showed normal hemodynamic parameters except for reduced numbers of eosinophils and the lack of eosinophilic response to parasites, which were also found in beta c mutant mice. The immune response of the beta c/IL-3 double-mutant mice to Listeria mono-cytogenes was normal, as was hematopoietic recovery after administration of the cytotoxic drug, 5-fluorouracil. Although it has been believed that IL-3/GM-CSF/IL-5 produced by activated T cells play a major role in expansion of hematopoietic cells in emergency, our results indicate that the entire function of IL-3/GM-CSF/IL-5 is dispensable for hematopoiesis in emergency as well as in the steady state. Thus, there must be an alternative mechanism to produce blood cells in both situations.

Perinatal lethality and blocked B-cell development in mice lacking the tyrosine kinase Syk.

The tyrosine kinase Syk (relative molecular mass 72,000), which is widely expressed in haematopoietic cells, becomes associated with and activated by engagement of the B-cell antigen receptor. Furthermore, it has been implicated in signalling through the receptors for interleukin-2 (IL-2), granulocyte colony-stimulating factor (G-CSF) and Fc, the T cell receptor, as well as through receptors for several platelet agonists. A homologous kinase, ZAP-70, is crucial in signalling through the T-cell receptor and in T-cell development. Using homologous recombination in embryonic stem cells, we created mice null for the syk gene which showed petechiae in utero and died shortly after birth. Irradiated mice reconstituted with Syk-deficient fetal liver showed a block in B-cell development at the pro-B to pre-B cell transition, consistent with a key role for Syk in pre-B-cell receptor signalling. Despite the production of small numbers of immature B cells, Syk-deficient radiation chimaeras failed to accumulate mature B cells, indicating a possible role for this protein in the production or maintenance of mature B cells. In addition, whereas the development of alpha beta T cells proceeded normally, Syk-deficient mice showed impaired development of thymocytes using the V gamma 3 variable region gene (V gamma 3+ thymocytes). Finally, we show that Syk is not required for signalling through the IL-2 and G-CSF receptors.

Defective antigen receptor-mediated proliferation of B and T cells in the absence of Vav.

Crosslinking of B- or T-cell antigen receptors results in the rapid tyrosine phosphorylation of a number of proteins, including Vav, a protein expressed in cells of the haematopoietic system. Vav contains an array of structural motifs that include Src-homology domains SH2/SH3 and regions of homology to the guanine-nucleotide-exchange protein Dbl, pleckstrin and protein kinase C (refs 5-9). Using the RAG-complementation approach, we have analysed in vivo differentiation and in vitro responses of B- and T-lineage cells generated by injection of embryonic stem cells homozygous for a null mutation in the vav gene into blastocysts of RAG-1- or RAG-2-deficient mice. Here we report that antigen receptor-mediated proliferative responses of B and T cells in vitro are severely reduced in the absence of Vav. We also suggest a direct link between the low proliferative response of Vav-deficient B and T cells and the reduced number of these cells in peripheral lymphoid organs of chimaeric mice.

Response of mouse skin tumors to doxorubicin is dependent on carcinogen exposure.

To investigate the role of carcinogenesis in determining the response of tumors to anticancer drugs, we have used the in vivo model of multistage carcinogenesis of the mouse skin. Mice were initiated with Harvey murine sarcoma virus or single and repeated applications of dimethylbenzanthracene (DMBA). The papillomas which developed as a result of these initiation protocols were monitored quantitatively for their response to the anticancer drug doxorubicin. A single dose of 10 mg/kg doxorubicin is relatively inefficient at reducing the frequency of papillomas arising as a result of either single or repeated applications of the chemical DMBA. However, virally initiated papillomas are sensitive to the single 10-mg/kg dose of doxorubicin and are reduced in frequency by greater than 80%. Repeat treatment with four doses of 5 mg/kg doxorubicin over a 4-week period also reveals differences in the responses of the papillomas to doxorubicin. As with the single dose of doxorubicin, papillomas initiated with multiple applications of DMBA showed only a limited response to four 5-mg/kg doses of doxorubicin. In comparison both the virally initiated and the single DMBA initiated papillomas responded to the four doses of doxorubicin and are reduced in frequency by about 80%. These data show that the response of papillomas to doxorubicin is related to the initiating event. Papillomas derived by viral initiation are most sensitive to doxorubicin while increasing the level of exposure to the chemical carcinogen DMBA increases the proportion of papillomas which do not respond to treatment with doxorubicin. There was no obvious relationship between the method of initiation or the treatment of the mice with doxorubicin and the levels of P-glycoprotein expression observed in the papillomas. All the papillomas expressed detectable levels of P-glycoprotein approaching that of the multidrug resistant cell line, CHRC5.

Construction and hormone regulation of a novel retroviral vector.

We report the analysis of a self-inactivating retroviral vector, constructed to allow inducible gene expression of inserted sequences from the mouse mammary tumour virus hormonal response element. The cloning strategy has been designed to allow for ease of insertion of the genes of interest. The vector contains the aph gene, allowing geneticin-resistance selection in mammalian cells. We have characterised dexamethasone (Dex)-induced increase in gene expression using the reporter gene encoding chloramphenicol acetyltransferase (CAT) inserted into the retroviral vector. We observe low basal levels of CAT activity in infected cells which is increased up to 50-fold by induction with Dex. The induction of pooled clones is 13.3-fold. Variation in Dex-induced CAT activity is observed in independently infected clones, which is not explained by proviral copy number.