Induction of angiogenesis during the transition from hyperplasia to neoplasia

It is now well established that unrestricted growth of tumours is dependent upon angiogenesis. Previous studies on tumour growth, however, have not revealed when or how the transition to an angiogenic state occurs during early tumour development. The advent of transgenic mice carrying oncogenes that reproducibly elicit tumours of specific cell types is providing a new format for studying multi-step tumorigenesis. In one of these models, transgenic mice expressing an oncogene in the beta-cells of the pancreatic islets heritably recapitulate a progression from normality to hyperplasia to neoplasia. We report here that angiogenic activity first appears in a subset of hyperplastic islets before the onset of tumour formation. A novel in vitro assay confirms that hyperplasia per se does not obligate angiogenesis. Rather, a few hyperplastic islets become angiogenic in vitro at a time when such islets are neovascularized in vivo and at a frequency that correlates closely with subsequent tumour incidence. These findings suggest that induction of angiogenesis is an important step in carcinogenesis.     

Progression from lymphoid hyperplasia to high-grade malignant lymphoma in mice transgenic for the t(14; 18)

Follicular lymphoma, the most common human lymphoma, characteristically has a t(14; 18) interchromosomal translocation. It is typically an indolent disease comprised of small resting B cells, but frequently develops into a high-grade lymphoma. The t(14; 18) translocates the Bcl-2 gene, generating a deregulated Bcl-2-immunoglobulin fusion gene. Bcl-2 is a novel inner mitochondrial membrane protein that extends the survival of certain cells by blocking programmed cell death. To determine the oncogenic potential of the t(14; 18) translocation, we produced transgenic mice bearing a Bcl-2-immunoglobulin minigene that structurally mimicked the t(14; 18). An indolent follicular hyperplasia in these transgenic mice progressed to a malignant diffuse large-cell lymphoma. The long latency, progression from polyclonal to monoclonal disease, and histological conversion, are all suggestive of secondary changes. Half of the immunoblastic high-grade lymphomas had a rearranged c-myc gene. Our transgenic mice provide an animal model for tumour progression in t(14; 18) lymphoma and show that prolonged B-cell life increases tumour incidence.     

Pre-B cells in kappa-transgenic mice

Recent experiments have shown that the microinjected kappa-chain gene of transgenic mice is expressed in a tissue-specific fashion only in B lymphocytes. The next step was to determine whether, within the B-lymphocyte lineage, the kappa-chain gene was expressed in a normal developmental fashion. Normally, only mu heavy(H)-chain genes, and not kappa-chain genes, are expressed in pre-B cells. To obtain cloned cell lines derived from early cells of the B-cell lineage, we transformed bone marrow cells from kappa-transgenic mice with Abelson murine leukaemia virus (A-MuLV) and tested the resultant cell lines for the retention of the kappa transgene and its expression in RNA and protein. We found that cells with the pre-B phenotype exist in kappa-transgenic mice. We further observed that in A-MuLV-transformed cell lines from a kappa-transgenic mouse with a high copy number of the transgene, the proportion of cell lines expressing kappa (transgenic kappa) was higher than in cell lines from normal or low copy number transgenic mice.     

Isotype exclusion and transgene down-regulation in immunoglobulin-lambda transgenic mice

A given B lymphocyte makes an antibody containing either kappa- or lambda-light chains, but not both. This isotype exclusion is effected at the level of the rearrangement of the immunoglobulin gene segments, although by an unknown mechanism. An attractive possibility is that, following productive rearrangement of one of the light-chain loci, the newly synthesized light-chain polypeptide inhibits DNA rearrangement for the other isotype. To test such feedback regulation, we have created transgenic mice carrying a rearranged lambda 1-gene. By contrast with the B cells in normal newborn mice which are mainly kappa+lambda-, the B cells in the newborn transgenic mice express lambda- but not kappa-chains. We propose that the synthesis of any light chain, be it kappa or lambda, that allows expression of IgM on the cell surface results in a cessation of all V-J joining. Interestingly, the limited light-chain repertoire of the transgenic mice does not persist and most adult B cells express endogenous kappa-rearrangements and down-regulate the transgene.     

Developmental regulation of human fetal-to-adult globin gene switching in transgenic mice

Transgenic mice containing a human fetal (gamma-) or adult (beta-) globin gene linked to the beta-globin gene locus activation region (LAR) express the gene throughout development. By contrast, transgenic mice containing LAR linked to both a fetal and an adult globin gene display the normal developmental switch from fetal to adult gene expression. This suggests that the human fetal-to-adult globin gene switch is controlled through a mutually exclusive interaction between LAR and either the gamma- or beta-globin gene, resulting in the expression of only one gene at any given moment.     

T-cell tolerance by clonal anergy in transgenic mice with nonlymphoid expression of MHC class II I-E

T-cell reactivity to the class II major histocompatibility complex I-E antigen is associated with T-cell antigen receptors containing the V beta gene segments V beta 17a and V beta 5. Mice expressing I-E with the normal tissue distribution (on B cells, macrophages, dendritic cells and thymic epithelium) induce tolerance to self I-E by clonal deletion in the thymus. By contrast, we find that transgenic INS-I-E mice that express I-E on pancreatic beta-cells, but not in the thymus or peripheral lymphoid organs, are tolerant to I-E but have not deleted V beta 5- and V beta 17a-bearing T cells. Moreover, whereas T-cell populations from nontransgenic mice proliferate in response to receptor crosslinking with V beta 5- and V beta 17a-specific antibodies, T cells from INS-I-E mice do not. Thus, our experiments provide direct evidence that T-cell tolerance by clonal paralysis does occur during normal T-cell development in vivo.     

Transgenic mice with inducible dwarfism

The pituitary gland, composed of the anterior, intermediate and posterior lobe, represents a principal regulatory interface through which the central nervous system controls body physiology. The ontogeny of the growth hormone (GH) and prolactin (Prl) producing cells of the anterior pituitary has been analysed in transgenic mice, using the thymidine kinase obliteration system (TKO). Cells expressing the herpes virus 1 thymidine kinase (HSV1-TK) gene acquire pharmacological sensitivity to synthetic nucleosides such as FIAU (1-(2-deoxy-2-fluoro-beta-delta-arabinofuranosyl)-5-iodouracil), whose metabolites kill dividing cells. Consequently we created transgenic mice carrying the HSV1-TK gene under the control of either the rat growth hormone or the rat prolactin promoter. If transgenic mice expressing HSV1-TK in somatotropes (GH-producing cells) are treated with FIAU, they develop as dwarfs. The anterior pituitary in these animals is nearly devoid of both somatotropes and lactotropes (Prl-producing cells). By contrast, transgenic mice expressing HSV1-TK in the lactotropes, treated with FIAU, have anatomically and histologically normal pituitaries. Because toxicity depends on cell division, we conclude that Prl expression and lactotrope differentiation are post-mitotic events. These results indicate that both somatotropes and lactotropes derive from a common GH-expressing stem-somatotrope. Unexpectedly, the stemsomatotrope is still present in the adult animal and is capable of repopulating the pituitaries of treated animals with mature GH and Prl producing cells.     

Depletion of the predominant B-cell population in immunoglobulin mu heavy-chain transgenic mice

The transgenic mouse line M54 was generated by introducing a functionally-rearranged immunoglobulin mu heavy-chain gene into the germ line of a C57B1/6 inbred mouse. Previous examination of the antibodies produced by B-cell hybridomas derived from transgenic M54 mice showed that the presence of the mu transgene grossly altered the immunoglobulin repertoire of unimmunized animals, suggesting that these mice suffer from a serious immunoregulatory perturbation. Studies presented here introduce a new perspective on this functional defect. We show that the lymphoid tissues from these transgenic mice lack virtually all conventional bone-marrow-derived B cells, which constitute the predominant B-cell population in normal mice and which typically produce primary and secondary antibody responses to T-cell-dependent antigens. Moreover, the bone marrow from transgenic M54 mice is depleted of pre-B lymphocytes, indicating a serious defect in early B-cell lymphopoiesis. In contrast, CD5 (Ly-1) B cells, a second B-cell population displaying a characteristic set of cell surface markers which are derived from distinct precursors in the peritoneum, are represented at normal frequencies in these transgenic mice. Thus, the presence of the rearranged immunoglobulin heavy-chain transgene in M54 mice results in an unexpected selective developmental defect that impairs the development of bone-marrow-derived pre-B and B cells without affecting Ly-1 B cells.     

Very high frequency of lymphoma induction by a chemical carcinogen in pim-1 transgenic mice

Infection of mice with Moloney murine leukaemia virus (MuLV) induces T-cell lymphomas after an average latency period of 150 days. In these lymphomas the MuLV DNA is frequently integrated into the mouse chromosomal DNA in the vicinity of the pim-1 oncogene. Transgenic mice overexpressing the pim-1 oncogene are predisposed to develop T-cell lymphomas, but only to the extent that approximately 10% of the mice develop a lymphoma within 240 days. When these mice are infected with MuLV, lymphomas develop in all mice in only 50-60 days. In these lymphomas MuLV DNA is integrated near either the c-myc or N-myc gene, suggesting that pim-1 and myc synergize in lymphomagenesis. To determine whether this system has a more general application, we have now tested the susceptibility of pim-1 transgenic mice to N-ethyl-N-nitrosourea (ENU), a chemical carcinogen. With a single low dose of ENU, nearly all pim-1 transgenic mice, but only 15% of non-transgenic mice, develop T-cell lymphomas within 200 days. All ENU-induced lymphomas in both pim-1 transgenic and non-transgenic mice express high levels of c-myc messenger RNA, supporting the notion that pim-1 and c-myc synergize in lymphoma induction. We propose that pim-1 transgenic mice could be used to test the oncogenic potential of other chemical compounds.     

转基因工程稻大米对小鼠的急性毒性与致突变研究

以转cry1Ac/sck双价抗虫基因工程稻MSA4、KF6-304、21S/MSB与II-32A/MSB大米为材料,进行了小鼠急性毒性试验、精子畸形试验与骨髓细胞微核试验研究,检测转基因稻大米对小鼠表观体症行为、脏器系数、血常规、精子畸形与微核率等的影响,以进一步评价转基因工程稻大米的食品安全性.小鼠急性毒性试验结果显示:转基因工程稻MSA4、KF6-304、21S/MSB与I-I-32A/MSB大米各剂量组灌喂小鼠,小鼠的行为、姿势、呼吸、毛色均正常;解剖观察其肝、脾、肾、心脏、胃、肠、生殖腺等主要脏器的外形、颜色、大小比例等无异常;血常规分析也显示受试鼠与对照材料组小鼠没有显著差异.小鼠精子畸形试验与骨髓细胞微核试验结果均显示转基因工程稻没有明显诱发小鼠精子畸变与骨髓细胞产生微核.其结果是:转基因工程稻MSA4、KF6-304、21S/MSB与II-32A/MSB大米未引起小鼠明显的急性毒性反应,对小鼠无明显的致畸与致突变作用.     

A role for clonal inactivation in T cell tolerance to Mls-1a

Clonal deletion plays a major part in the maintenance of natural self-tolerance in both normal and transgenic mice. Self antigens that are expressed in the thymus result in the physical elimination of autoreactive thymocytes at a particular stage in their development. For example, the majority V beta 6- and V beta 8.1-bearing T cells that recognize the minor lymphocyte-stimulating antigen, Mls-1a (ref. 10) , are clonally deleted in the thymuses of normal mice and transgenic mice expressing Mls-1a (refs 2, 3, 9). In contrast, a very different mechanism of tolerance involving the functional inactivation, but not elimination, of autoreactive cells, termed clonal inactivation or clonal anergy, has been implicated in some experimentally manipulated systems of tolerance. To test further the mechanisms involved in self-tolerance, we have generated transgenic mice expressing a V beta 8.1 beta chain on greater than 95% of peripheral T cells and have tested tolerance to Mls-1a in these mice. Surprisingly, a significant fraction of the CD4+ peripheral cells that survived deletion were non-responsive in vitro to any stimulus tested. Naturally occurring tolerance to a self antigen expressed in the thymus can thus be mediated by clonal anergy, as well as by clonal deletion.     

Spexin转基因小鼠的构建及表型初步分析

Spexin是一种具有多种生物学功能的神经肽,在代谢疾病及神经系统疾病中发挥重要作用.为了深入研究spexin的生理功能和相关机制,构建了spexin转基因小鼠,通过检测血液中spexin含量以及糖耐受实验,对模型小鼠进行了初步的表型分析.利用piggyBac(PB)转座子技术培育spexin转基因小鼠,采用聚合酶链反应(polymerase chain reaction,PCR)和酶联免疫吸附测定法(enzyme-linked immunosorbent assay,ELISA)分别在基因层面和蛋白层面验证了模型动物是否构建成功.之后用饲喂高脂饲料的雄性小鼠进行糖耐受实验,对转基因模型小鼠进行表型分析.PCR结果显示,以靶向spexin的两对引物扩增,转基因型小鼠基因组DNA中能分别扩增出300 bp和301 bp的片段,而野生型小鼠则不能.ELISA结果中,转基因型小鼠血清中spexin含量较野生型小鼠显著升高;同一表型中,雌雄小鼠间血清中spexin水平无明显差异.饲喂高脂饲料85 d内,转基因型小鼠和野生型小鼠体重变化未观察到显著差异;糖耐受结果显示,转基因型小鼠每个时间点血糖浓度均低于野生型小鼠,且在15 min时具有显著性差异.结果显示成功构建了过表达spexin基因的小鼠模型.spexin表达水平的升高在一定程度上提高了C57BL/6小鼠的糖耐受能力.该模型为进一步研究spexin基因在动物体内的功能以及在代谢疾病发病机制中的作用奠定了基础.     

Expression of human growth hormone-releasing factor in transgenic mice results in increased somatic growth

The neurohumoral regulation of growth hormone secretion is mediated in part by two hypothalamic peptides that reach the anterior pituitary via the hypothalamo-hypophysial portal blood system. Somatostatin inhibits the release of growth hormone, whereas growth hormone-releasing factor (GRF) positively regulates both growth hormone synthesis and secretion. Two forms of human GRF, 40 and 44 amino acids long, have been characterized from extra-hypothalamic tumours as well as from the hypothalamus. Analysis of human GRF complementary DNA and genomic clones indicates that the GRF peptides are first synthesized as a 107- or 108-amino-acid precursor protein. To examine the physiological consequences of GRF expression, we have established strains of transgenic mice containing a fusion gene including the promoter/regulatory region of the mouse metallothionein-I (MT-I) gene and the coding region of the human GRF gene. We report that expression of the human GRF precursor protein in these animals results in measurable levels of human GRF and increased levels of mouse growth hormone in plasma and accelerated growth rates relative to control littermates. These results demonstrate a direct role for GRF in the positive regulation of somatic growth. Unexpectedly, female transgenic mice carrying the MT-GRF fusion gene are fertile, in contrast to female transgenic mice expressing human or rat growth hormone, which are generally infertile. These transgenic mouse strains should provide useful animal models for the study of several types of human growth disorders.     

Site-independent expression of the chicken beta A-globin gene in transgenic mice

The level of expression of exogenous genes carried by transgenic mice typically varies from mouse to mouse and can be quite low. This behaviour is attributed to the influence of the mouse chromatin near the site of transgene integration. This 'position effect' has been seen in transgenic mice carrying the human beta-globin gene. It was however, abolished when DNase I hypersensitive sites (normally found 65 to 44 kilobases (kb) upstream) were linked to the human beta-globin transgene. Thus, the upstream DNA (previously named a dominant control or locus activation region, now denoted a locus control region) conferred the ability to express human beta-globin at high levels dependent on copy number on every mouse carrying the construct. We report here an investigation of chicken beta A-globin gene expression in transgenic mice. A 4.5-kb fragment carrying the beta A-globin gene and its downstream enhancer, without any far upstream elements, is sufficient to ensure that every transgenic mouse expresses chicken globin messenger RNA at levels proportional to the transgene copy number. Thus the chicken DNA elements that allow position-independent expression can function in mice. In marked contrast to the human beta cluster, these elements are no farther than 2 kb from the gene. The location of the elements within the cluster demonstrates that position independence can be mediated by DNA that does not define a gene cluster boundary.     

Self-tolerance to transgenic gamma delta T cells by intrathymic inactivation

During their intrathymic differentiation, T lymphocytes expressing alpha beta T-cell receptors (TCR) are negatively and positively selected. This selection contributes to the establishment of self-tolerance and ensures that mature CD4+ and CD8+ cell populations are restricted by the self major histocompatibility complex. Little is known, however, about gamma delta T-cell development. To investigate whether selection operates in the establishment of the gamma delta T-cell class, we have generated transgenic mice using gamma- and delta-transgenes encoding a TCR that is specific for a product of a gene in the TL-region of the TLb haplotype. Similar numbers of thymocytes expressing the transgenic TCR were generated in mice of TLb and TLd haplotypes. But gamma delta thymocytes from TLb and TLd transgenic mice differed in cell size, TCR density and in their capacity to respond to TLb stimulator cells or interleukin-2 (IL-2). In contrast to gamma delta T cells from TLd transgenic mice, gamma delta T cells from TLb transgenic mice did not produce IL-2 and did not proliferate in response to TLb stimulator cells, but they did proliferate in the presence of exogenous IL-2. These results indicate that functional inactivation of self-antigen-specific T cells could contribute to the establishment of self-tolerance to thymic determinants.