Somatic hypermutation of an immunoglobulin transgene in kappa transgenic mice

Initial studies of somatically acquired mutations in immunoglobulin V regions from hybridomas and myelomas that are not derived from joining aberrations, suggested a controlled and specific hypermutation process, because spontaneous mutation rates observed for other genes are extremely low. Some evidence for the idea that mutations are introduced during V-gene rearrangement came from the clustering of mutations at the joining sites, from the absence of mutations in unrearranged V genes and from the low level of mutations in only partially (D-J) rearranged nonproductive heavy-chain alleles. Another model in which mutations accumulate with each cell division, rather than being introduced all at once, was supported by the finding that immunoglobulin genes of hybridomas derived from a single mouse frequently had several mutations in common, and so might be derived from the same precursor cell whose daughters then accumulated additional mutations. But the common mutations in some cases could be due to as yet unidentified related germline genes, or could represent the effect of antigen selection for certain amino acids. To try to detect hypermutation in the absence of V-gene rearrangement, we isolated B lymphocytes with endogenous heavy-chain gene mutations from transgenic mice carrying pre-rearranged kappa-transgenes. We found that these kappa-transgenes were also somatically mutated. This and other observations indicated that: ongoing rearrangement is not required for mutation; there are signals for hypermutation in the transgenes; the mutations are found only in the variable region, so the constant region may not be a target; different transgene insertion sites are compatible with hypermutations and more than one transgene is expressed in the same cell.     

Can transgenic rice cause ecological risks through transgene escape?

Alien transgene escape from genetically engineered rice to non-transgenic varieties or close wild relatives (including weedy rice) may lead to unpredictable ecological risks. However, for transgene escape to occur three conditions need to be met: (i) spatially, transgenic rice and its non-transgenic counterparts or wild relatives should have sympatric distributions; (ii) temporally, the flowering time of transgenic rice and the non-transgenic varieties or wild relatives should overlap; and (iii) biologically, transgenic rice and its wild relative species should have such a sufficiently close relationship that their interspecific hybrids can have normal generative reproduction. This paper presents research data on the geographic distribution, flowering habits, interspecific hybridization, and gene flow of cultivated rice (Oryza sativa) and its closely related wild relatives containing the AA genome. The objective is to estimate the possibility of transgene escape to non-transgenic rice varieties and wild relatives of rice, which may result in unpredictable ecological risks.     

Application of a nuclear localization signal gene in transgene mice

Efficient gene transfer by cytoplasm co-injection will offer a powerful means for transgenic animals. Using co-injection in cytoplasm, two independent gene constructs, including bovine α-s1-casein-hG-CSF and a mammal expression vector expressing a nuclear localization signal (mNLS), were introduced into fertilized mouse eggs. The target gene construct was docked into host nucleus probably by the nuclear localization signal. Transgene mice have been obtained at 58% (29/50) of integration ratio. Expression level of the positive transgene mice was detected by Western blotting. Maximal expression of human G-CSF was estimated about 540 mg/L of milk. The expression ratio was up to 75% (9/12). The results here have important practical implications for the generation of mammary gland bioreactors and other transgene studies. Co-injection of a target gene with an expression vector of a mammal nuclear localization signal by cytoplasm appears to be a useful, efficient and easy strategy for generating transgenic animals, which may be able to substitute the routine method of pronucleus-injection of fertilized eggs.     

两种转基因小鼠生长繁殖方法的研究

目的：对两种转基因小鼠的生长繁殖方法进行研究。方法：在SPF环境下,用C57BL／6小鼠做对照,采取近亲交配的繁殖方法研究其生长繁殖性能,并检测两种转基因小鼠体内荧光蛋白的表达。结果：两种转基因小鼠的生长繁殖性能均与C57BL／6小鼠相一致,所有脏器重量和绝大多数正常血液生化指标与C57BL／6小鼠无显著性差异（P〉0．05）;两种转基因小鼠脑片检测可见在不同的年龄段均有相应荧光蛋白的表达。结论：在SPF环境下采用近亲交配繁殖法培育转基因小鼠是成功的。     

Pituitary hyperplasia and gigantism in mice caused by a cholera toxin transgene.

Cyclic AMP is thought to act as an intracellular second messenger, mediating the physiological response of many cell types to extracellular signals. In the pituitary, growth hormone (GH)-producing cells (somatotrophs) proliferate and produce GH in response to hypothalamic GH-releasing factor, which binds a receptor that stimulates Gs protein activation of adenylyl cyclase. We have now determined whether somatotroph proliferation and GH production are stimulated by cAMP alone, or require concurrent, non-Gs-mediated induction of other regulatory molecules by designing a transgene to induce chronic supraphysiological concentrations of cAMP in somatotrophs. The rat GH promoter was used to express an intracellular form of cholera toxin, a non-cytotoxic and irreversible activator of Gs. Introduction of this transgene into mice caused gigantism, elevated serum GH levels, somatotroph proliferation and pituitary hyperplasia. These results support the direct triggering of these events by cAMP, and illustrate the utility of cholera toxin transgenes as a tool for physiological engineering.     

Obtaining the transgenic poplars with low lignin content through down-regulation of 4CL

The antisense 4CL (4-coumarate: CoA ligase)gene was transformed into triploid Chinese white poplar(Populus tomentosa) mediated by Agrobacterium tumefaciens.PCR and Southern blot analysis indicated that antisense 4CLgene had been integrated into the genome of the transgenicChinese white poplars. The antisense gene had also beenexpressed, which was indicated by RT-PCR and Westernanalysis. Klason lignin content assay showed that repressionof 4CL expression could result in remarkable reduction oflignin content in transgenic poplars, with most reduction of 41.73% compared with that of wild type in this paper. Butthere is no significant difference in holocellulose content be-tween trans- genic and wild poplars. We considered that 4CLmight not be the metabolism control point between ligninand carbohy- drate biosynthesis. The stems of transgenicpoplars displayed red-brown color with different levels afterthe bark was peeled, while those of untransformed plantswere white. No visible differences in growth and developmentwere observed between transgenic and wild plants. Wiesnerreaction analysis of the transgenic plant stems with reducedlignin content exhibited red color, while that of untrans-formed plant was typically purple-red.     

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.     

Impaired type II glucocorticoid-receptor function in mice bearing antisense RNA transgene.

Glucocorticoids, in conjunction with their cognate receptors, exert negative-feedback effects on the hypothalamus-pituitary-adrenal axis, suppressing adrenal steroid secretions. Two types of corticosteroid receptor, distinguishable by their ability to bind corticosterone, have been identified as classical mineralocorticoid (type I) and glucocorticoid (type II) receptors by cloning their complementary DNAs. The type I receptor controls the basal circadian rhythm of corticosteroid secretion. Both receptor types are involved in negative feedback, but the type II receptor may be more important for terminating the stress response as it is the only one to be increased in animals rendered more sensitive to corticosteroid negative-feedback effects. Here we create a transgenic mouse with impaired corticosteroid-receptor function by partially knocking out gene expression with type II glucocorticoid receptor antisense RNA. We use this animal to study the glucocorticoid feedback effect on the hypothalamus-pituitary-adrenal axis.     

Bovine papillomavirus genome elicits skin tumours in transgenic mice

Transmission of the bovine papillomavirus-1 (BPV-1) genome through the mouse germ line results in the heritable formation of fibropapillomas of the skin, a tissue-specific phenotype analogous to that observed in natural BPV-1 infection of cattle. Oncogenesis is slow, with tumours first arising at 8-9 months of age, usually in areas prone to wounding. Extrachromosomal BPV-1 DNA is detected in all tumours, whereas normal tissues show only integrated DNA.     

Prevention of diabetes in non-obese diabetic I-Ak transgenic mice

The non-obese diabetic (NOD) mouse develops insulin-dependent diabetes mellitus (IDDM) with mononuclear cell infiltration of the islets of Langerhans and selective destruction of the insulin-producing beta-cells, as in humans. Most infiltrating cells are T lymphocytes, and most of these carry the CD4 antigen. Adoptive transfer of T cells from diabetic NOD mice into irradiated NOD or athymic nude NOD mice induces diabetes. Susceptibility to IDDM in NOD mice is polygenic, with one gene linked to the major histocompatibility complex class II locus, which in NOD mice expresses a unique I-A molecule but no I-E. Speculation exists as to the role of the I-A molecule in the diabetes susceptibility of NOD mice, especially regarding the significance of specific unique residues. To examine the role of the NOD I-A molecule in IDDM pathogenesis, we made NOD/Lt mice transgenic for I-Ak by microinjecting I-Ak alpha- and beta-genes into fertilized NOD/Lt eggs. Insulitis was markedly reduced and diabetes prevented in NOD/Lt mice expressing I-Ak.     

HLA-restricted recognition of viral antigens in HLA transgenic mice

Cytotoxic T lymphocytes (CTL) recognize antigen in the context of the class-I products of the major histocompatibility complex (MHC). The extensive polymorphism of class-I molecules is thought to be linked to their capacity to present a large variety of foreign antigens. Whether a single T-cell receptor (TCR) recognizes two separate epitopes (the foreign antigen and an epitope on MHC molecules), or a single epitope resulting from the combination of a foreign antigen and an MHC molecule, has not yet been resolved. In view of the differences between species in primary structure of histocompatibility antigens, it might be predicted that the TCR repertoire would evolve in concert with the diversity of MHC antigens. The mouse and human TCR repertoire would be optimally adapted to engage in productive interactions only with mouse (H-2) and human (HLA) MHC antigens respectively, especially if the more conserved features of histocompatibility antigens, in addition to foreign antigen, were seen by the TCR. Alternatively, only the most variable segments of MHC antigens might be engaged in antigen presentation and thus in interaction with the TCR. In that case, interaction between MHC plus antigen and the TCR might not necessarily be limited by species-specific features. By analysis of the T-cell response against virus-infected cells in HLA-B27/human beta 2-microglobulin double transgenic mice, we report here that the mouse T-cell repertoire is perfectly capable of using the human HLA-B27 antigen as a restriction element.     

Regulation of human insulin gene expression in transgenic mice

Insulin is a polypeptide hormone of major physiological importance in the regulation of fuel homeostasis in animals (reviewed in refs 1,2). It is synthesized by the beta-cells of pancreatic islets, and circulating insulin levels are regulated by several small molecules, notably glucose, amino acids, fatty acids and certain pharmacological agents. Insulin consists of two polypeptide chains (A and B, linked by disulphide bonds) that are derived from the proteolytic cleavage of proinsulin, generating equimolar amounts of the mature insulin and a connecting peptide (C-peptide). Humans, like most vertebrates, contain one proinsulin gene, although several species, including mice and rats, have two highly homologous insulin genes. We have studied the regulation of serum insulin levels and of insulin gene expression by generating a series of transgenic mice containing the human insulin gene. We report here that the human insulin gene is expressed in a tissue-specific manner in the islets of these transgenic mice, and that serum human insulin levels are properly regulated by glucose, amino acids and tolbutamide, an oral hypoglycaemic agent.     

Peripheral neuropathies, hepatocellular carcinomas and islet cell adenomas in transgenic mice

The ability to introduce foreign DNA into the genome of mice offers unique opportunities to produce new models of disease process. Recent experiments have shown that integration and expression of simian virus 40 (SV40) T antigen genes and the murine mammary tumour virus (MMTV)-myc genes in transgenic mice can lead to the development of neoplasia in a remarkably tissue-specific manner. In the case of SV40-bearing mice, tumours consistently develop in the choroid plexus. In the accompanying paper, we show that the 72-base pair (bp) enhancer in the SV40 genome is instrumental in directing tumorigenesis to the choroid plexus. However, when the enhancer is deleted from a construction also containing the metallothionein-human growth hormone fusion gene (SV delta e-MGH), an entirely new pattern of pathology results. The present report focuses on transgenic mice carrying this construct; they develop demyelinating peripheral neuropathies, hepatocellular carcinomas and islet cell adenomas.     

Genomic imprinting determines methylation of parental alleles in transgenic mice

Mouse embryogenesis relies on the presence of both the maternal and the paternal genome for development to term. It has been proposed that specific modifications are imprinted onto the chromosomes during gametogenesis; these modifications are stably propagated, and their expression results in distinct and complementary contributions of the two parental genomes to the development of the embryo and the extraembryonic membranes. Genetic data further suggest that a substantial proportion of the genome could be subject to chromosomal imprinting, the molecular nature of which is unknown. We used random DNA insertions in transgenic mice to probe the genome for modified regions. The DNA methylation patterns of transgenic alleles were compared after transmission from mother or father in seven mouse strains carrying autosomal insertions of the same transgenic marker. One of these loci showed a clear difference in DNA methylation specific for its parental origin, with the paternally inherited copy being relatively undermethylated. This difference was observed in embryos on day 10 of gestation, but not in their extraembryonic membranes. Moreover, the methylation pattern was faithfully reversed upon each germline transmission to the opposite sex. Our findings provide evidence for heritable molecular differences between maternally and paternally derived alleles on mouse chromosomes.     

Reduction of choline acetyltransferase activities in APP770 transgenic mice

Transgenic mice overexpressing the 770-amino acid isoform of human Alzheimer amyloid precursor protein exhibit extracellular b -amyloid deposits in brain regions including cerebral cortex and hippocampus, which are severely affected in Alzheimer's disease patients. Significant reduction in choline acetyltransferase (ChAT) activities has been observed in both cortical and hippocampal brain regions in the transgenic mice at the age of 10 months compared with the age-matched non-transgenic mice, but such changes have not been observed in any brain regions of the transgenic mice under the age of 5 months. These results suggest that deposition of b -amyloid can induce changes in the brain cholinergic system of the transgenic mice.     

Formation of beta-amyloid protein deposits in brains of transgenic mice

Deposits of beta-amyloid are one of the main pathological characteristics of Alzheimer's disease. The beta-amyloid peptide constituent (relative molecular mass 4,200) of the deposits is derived from the beta-amyloid precursor protein (beta-APP) which is expressed in several different isoforms. The two most prevalent beta-APP isoforms are distinguished by either the presence (beta-APP751) or absence (beta-APP695) of a Kunitz serine protease inhibitor domain. Changes in the abundance of different beta-APP messenger RNAs in brains of Alzheimer's disease victims have been widely reported. Although these results have been controversial, most evidence favours an increase in the mRNAs encoding protease inhibitor-containing isoforms of beta-APP and it is proposed that this change contributes to beta-amyloid formation. We have now produced an imbalance in the normal neuronal ratio of beta-APP isoforms by preparing transgenic mice expressing additional beta-APP751 under the control of a neural-specific promoter. The cortical and hippocampal brain regions of the transgenic mice display extracellular beta-amyloid immunoreactive deposits varying in size (less than 5-50 microns) and abundance. These results suggest that one mechanism of beta-amyloid formation may involve a disruption of the normal ratio of neuronal beta-APP isoform expression and support a direct relationship between increased expression of Kunitz inhibitor-bearing beta-APP isoforms and beta-amyloid deposition.