Heat shock proteins are proposed to limit injury resulting from diverse environmental stresses, but direct metabolic evidence for such a cytoprotective function in vertebrates has been largely limited to studies of cultured cells. We generated lines of transgenic mice to express human 70-kDa heat shock protein constitutively in the myocardium. Hearts isolated from these animals demonstrated enhanced recovery of high energy phosphate stores and correction of metabolic acidosis following brief periods of global ischemia sufficient to induce sustained abnormalities of these variables in hearts from nontransgenic littermates. These data demonstrate a direct cardioprotective effect of 70-kDa heat shock protein to enhance postischemic recovery of the intact heart.Previous studies have demonstrated a cytoprotective function for heat shock proteins during thermal stress or energy deprivation in cultured cells (1-6). Expression of endogenous 70-kDa heat shock protein (hsp7o) is induced by ischemia in intact hearts (6, 7), and this response correlates with a reduced susceptibility to ischemic injury (8-11). Preconditioning stimuli that induce hsp70, however, also invoke other responses that may mitigate myocardial dysfunction resulting from ischemia (12)(13)(14). The proposition that stress proteins exert direct cardioprotective effects remained conjectural until the development of transgenic mouse models, which provide an opportunity for rigorous tests of this hypothesis by examination of relationships between heat shock protein expression and various descriptors of ischemic injury. The goal of this work was to assess the effects of forced constitutive expression of hsp70 on recovery of high-energy phosphate metabolism (assessed by 31P NMR spectroscopy) following periods of global ischemia in isolated perfused mouse hearts. assay. RNA was extracted from myocardial tissue samples by standard methods, and cDNA was synthesized with reverse transcriptase by using an oligo(dT) primer. Primer pairs were specific either for murine glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (loading control) or human hsp7o cDNA and generated amplification products of 400 or 496 bp, respectively. RNA isolated from heat shocked human HeLa cells served as a positive control. The identity of the PCR product generated from the transgenic hearts when using human hsp7o primer pairs was confirmed by cleavage with Cla I, the restriction site for which is absent from the murine hsp70 sequence, to produce a 453-bp fragment. Proteins extracted from whole hearts were separated by isoelectric focusing and SDS/PAGE, and two-dimensional immunoblot assays (17) were conducted by using an exquisitely sensitive rabbit antihuman inducible hsp7o antibody. MATERIALS AND METHODSLangendorff Perfusion. The care and treatment of all animals in this study were in accord with the recommendations of the National Institutes of Health and the U. S. Department of Health and Human Services. The protocol was approved by the Institutional Review Board for Animal Rese...

SummaryTo investigate the role of interleukin 7 (IL-7) in the development of the lymphoid system, we have generated two lines of transgenic mice carrying an Ib7 eDNA fused to an immunoglobulin heavy chain promoter and enhancer. This transgene is expressed in the bone marrow, lymph nodes, spleen, thymus, and skin provoking a perturbation of T cell development characterized by a marked reduction of CD4+CD8 + (double-positive) thymocytes. Quite unexpectedly, however, both lines also develop a progressive cutaneous disorder involving a dermal lymphoid infiltrate that results in progressive alopecia, hyperkeratosis, and exfoliation. Although the infiltrate is primarily composed of T lineage cells, its development is not impeded in the athymic nu/nu background. Furthermore, the phenotype can be transmitted horizontally by transplanting lymphoid tissues or skin to syngeneic wild-type mice. Thus, the phenotype is conveyed by skin-homing, mobile cells (presumably the infiltrating lymphocytes) in a cell-autonomous fashion. In addition to the skin phenotype, this transgene also provokes the development of a lymphoproliferative disorder that induces B and T cell lymphomas within the first 4 mo of life. These findings suggest potential physiologic actions of IL-7 in T cell development and in cutaneous immunity. They also demonstrate that IL-7 can act as an oncogene in the living organism.

We have developed a binary transgenic system that activates an otherwise silent transgene in the progeny of a simple genetic cross. The system consists of two types of transgenic mouse strains, targets and transactivators. A target strain bears a transgene controlled by yeast regulatory sequences (UAS) that respond only to the yeast transcriptional activator GALA. A transactivator strain expresses an active

T lymphocytes have been implicated in the onset of many autoimmune diseases; however, the mechanism underlying T-cell activation toward self antigens are poorly understood. To study whether T-lymphocyte costimulation can overcome the immunologic unresponsiveness observed in an in Wivo model, we have created tansgenic mice essing the costimulatory mouse molecule B7-1, a lignd for the CD28 receptor, on pancreatic beta cells. We now report that tripletransgenic mice expressing both B7-1 and a viral glycoprotein on their beta cells, along with T cells expressing the viralglycoprotein-speifc ransgenc T-cell receptor, all develop insulitis (ymphocytic infiltration of the pancreatic islets) and diabetes. In striking contrast, the T cells in double-tansgenic mice expressing the same viral glycoprotein (but no B7) on their pancreatic beta cells and the traenic T-cell receptor on their T cells, reported earlier, remain indifferent to the glycoproteinexpressing beta cells. In fact, all three transgenes are required to initiate immune-mediated destruction of the beta cells. Mice expressing any of the transgenes alone, or any two in combination, maintain normal islet architecture and never spontaneously develop insulitis or diabetes. Our results show that aberrant B7 expression on peripheral tissues may play an important role in the activation of self-reactive T cells and further suggest that abnormal expression of costimulatory receptors may be involved in various T-cell-mediated autoimmune diseases.

Mammalian spermatogenesis is a complex developmental process. The analysis of mouse mutations has provided insight into biochemical pathways required for completion of this process. We previously described the autosomal recessive mouse morc TgN(Tyr)1Az(microrchidia) mutation, a serendipitous transgenic insertional mutation which causes arrest of spermatogenesis prior to the pachytene stage of meiosis prophase I. We now report the molecular characterization of the morc locus and positional cloning of a gene disrupted by the morc TgN(Tyr)1Az mutation. This gene, which we term Morc, encodes a 108 kDa protein expressed specifically in male germ cells. The transgene integrated within the first intron of Morc and was accompanied by an intragenic deletion of approximately 13 kb of genomic sequences, removing exons 2-4 and abrogating expression of the wild-type transcript. Analysis of the MORC protein sequence revealed putative nuclear localization signals, two predicted coiled-coil structural motifs and limited homology to GHL (GyraseB, Hsp90, MutL) ATPase. Epitope-tagged MORC protein expressed in COS7 cells localized to the nucleus. We also cloned the human MORC homolog and show that it too is testis-specific, but closely related human genes are transcribed in multiple somatic tissues. Homologous proteins are also present in zebrafish, nematodes, slime mold and plants. Thus, cloning of Morc defines a novel gene family whose members are likely to serve important biological functions in both meiotic and mitotic cells of multicellular organisms.

We have derived putative embryonic stem (ES) cell lines from preimplantation rabbit embryos and report here their initial characterization. Two principal cell types emerged following serial passage of explanted embryos, and each has subsequently given rise to immortalized cell lines. One cell type has morphology identical to primary outgrowths of trophectoderm, is strictly feeder-cell dependent, and spontaneously forms trophectodermal vesicles at high cell density. The second type appears to represent pluripotent ES cells derived from the inner cell mass as evidenced by 1) ability to grow in an undifferentiated state on feeder layers, 2) maintenance of a predominantly normal karyotype through serial passage (over 1 year), and 3) ability to form embryoid bodies, which form terminally differentiated cell types representative of ectoderm, mesoderm, and endoderm. These ES cells may ultimately be suitable for introduction of germline mutations (via homologous recombination). The rabbit's size, reproductive capability, and well-characterized physiology make it suitable for a wide range of investigations, particularly for development of large animal models of human disease.

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Genetically altered mice may exhibit highly variable phenotypes due to the variation in genetic background, which can only be circumvented by generation of inbred, isogenic gene-targeted and control mice. Here we report that an embryonic stem (ES) cell culture medium conditioned by a rabbit fibroblast cell line transduced with genomic rabbit leukemia inhibitory factor allows efficient derivation and maintenance of ES cell lines from all of 10 inbred mouse strains tested, including some that were presumed to be nonpermissive for ES cell derivation (