Synthesis of functional human hemoglobin in transgenic mice

Human alpha- and beta-globin genes were separately fused downstream of two erythroid-specific deoxyribonuclease (DNase) I super-hypersensitive sites that are normally located 50 kilobases upstream of the human beta-globin gene. These two constructs were coinjected into fertilized mouse eggs, and expression was analyzed in transgenic animals that developed. Mice that had intact copies of the transgenes expressed high levels of correctly initiated human alpha- and beta-globin messenger RNA specifically in erythroid tissue. An authentic human hemoglobin was formed in adult erythrocytes that when purified had an oxygen equilibrium curve identical to the curve of native human hemoglobin A (Hb A). Thus, functional human hemoglobin can be synthesized in transgenic mice. This provides a foundation for production of mouse models of human hemoglobinopathies such as sickle cell disease.     

Correction of sickle cell disease in adult mice by interference with fetal hemoglobin silencing

Persistence of human fetal hemoglobin (HbF, α(2)γ(2)) in adults lessens the severity of sickle cell disease (SCD) and the β-thalassemias. Here, we show that the repressor BCL11A is required in vivo for silencing of γ-globin expression in adult animals, yet dispensable for red cell production. BCL11A serves as a barrier to HbF reactivation by known HbF inducing agents. In a proof-of-principle test of BCL11A as a potential therapeutic target, we demonstrate that inactivation of BCL11A in SCD transgenic mice corrects the hematologic and pathologic defects associated with SCD through high-level pancellular HbF induction. Thus, interference with HbF silencing by manipulation of a single target protein is sufficient to reverse SCD.     

Gelation of sickle cell hemoglobin: effects of hybrid tetramer formation in hemoglobin mixtures

The altered gelation behavior found in mixtures of sickle cell hemoglobin with other hemoglobins is due to the formation of hybrid hemoglobin tetramers from unlike dimers. The hemoglobins need not possess the deoxy quaternary structure for gelation to occur; liganded forms are also capable of participation in gelation.     

Fetal hemoglobin variants in mice

Two strains of mice, DBA and C3H, have a fetal globin polypeptide chain which differs in electrophoretic mobility from the corresponding fetal chain of the C57B1 strain. Mice of the DBA and C3H strains also differ from those of the C57B1 in adult hemoglobin type. Results of backcrossing the (DBA x C57B1) hybrid to the C57B1 suggest that the fetal chain locus and the adult beta-chain locus are closely linked.     

Oncogenesis of the lens in transgenic mice

Neoplastic tumors of the ocular lens of vertebrates do not naturally occur. Transgenic mice carrying a hybrid gene comprising the murine alpha A-crystallin promoter (-366 to +46) fused to the coding sequence of the SV40 T antigens developed lens tumors, which obliterated the eye cavity and even invaded neighboring tissue, thus establishing that the lens is not refractive to oncogenesis. Large-T antigen was detected early in lens development; it elicited morphological changes and specifically interfered with differentiation of lens fiber cells. Both alpha- and beta-crystallins persisted in many of the lens tumor cells, while gamma-crystallin was selectively reduced. Accessibility, characteristic morphology, and defined protein markers make this transparent epithelial eye tissue a potentially useful system for testing tumorigenicity of oncogenes and for studying malignant transformation from its inception until death of the animal.     

透明颤菌血红蛋白基因的导入对黄原胶合成的影响

为研究透明颤菌血红蛋白基因vgb对黄原胶合成的影响,采用原生质体电转化的方法,将vgb与质粒pUC18构建的重组克隆pUC-LV,转入野油菜黄单胞杆菌中,并对转化菌和初发菌进行了PCR检测和摇瓶发酵试验。结果显示:vgb成功导入黄单胞杆菌,与初发菌相比,转化菌的黄原胶产量平均提高了15.91%,说明透明颤菌血红蛋白基因vgb的导入有利于黄原胶的合成。     

Polymorphism of sickle cell hemoglobin aggregates: structural basis for limited radial growth

Fibers composed of molecules of deoxygenated sickle cell hemoglobin are the basic cause of pathology in sickle cell disease. The hemoglobin molecules in these fibers are arranged in double strands that twist around one another with a long axial repeat. These fibrous aggregates exhibit a pattern of polymorphism in which the ratio of their helical pitch to their radius is approximately constant. The observed ratio agrees with an estimate of its value calculated from the geometric properties of helical assemblies and the degree of distortion that a protein-protein interface can undergo. This agreement indicates that the radius of an aggregate is limited by the maximum possible stretching of double strands. The geometric properties limiting the radial extent of sickle hemoglobin fibers are fundamental to all cables of protein filaments and could contribute to the control of diameter in other biological fibers such as collagen or fibrin.     

诱导性免疫共刺激分子转基因小鼠模型的建立

利用RT—PCR方法从人扁桃体的激活T细胞mRNA中扩增出诱导性免疫共刺激分子(ICOS)cDNA,继而将ICOS基因插入到pEGFP—N3中得到含ICOS和GFP融合蛋白的pEGFP—ICOS。将外源基因pEGF、P—ICOS注射至FVB小鼠原核中,其胚胎移植到同期发情的假孕受体产出后代,经PCR和Southern检测获得阳性转基因小鼠。试验共注射移植胚胎117枚,出生小鼠43只,检出阳性小鼠2只,该转基因已稳定遗传至F3代,说明建立了ICOS转基因小鼠模型。     

转β-甘露聚糖酶基因manA小鼠的制备

为了获得β-甘露聚糖酶基因manA的转基因小鼠,从黑曲霉中克隆得到β-甘露聚糖酶基因manA,进行体外表达检测甘露聚糖酶活性后,将此基因插入到含有猪腮腺分泌蛋白(PSP)基因启动子的表达载体pPSPBGPneo中,得到在腮腺组织特异表达β-甘露聚糖酶基因manA的载体pPSP-manA,总长为16.3 kb,将其进行线性化后回收得到高质量DNA片段,通过显微注射得到17只原代小鼠,进行PCR和Southern blot检测发现有6只阳性转基因小鼠,表明转β-甘露聚糖酶基因manA小鼠制备成功.     

Spontaneous neurodegeneration in transgenic mice with mutant prion protein

Transgenic mice were created to assess genetic linkage between Gerstmann-Str?ussler-Scheinker syndrome and a leucine substitution at codon 102 of the human prion protein gene. Spontaneous neurologic disease with spongiform degeneration and gliosis similar to that in mouse scrapie developed at a mean age of 166 days in 35 mice expressing mouse prion protein with the leucine substitution. Thus, many of the clinical and pathological features of Gerstmann-Str?ussler-Scheinker syndrome are reproduced in transgenic mice containing a prion protein with a single amino acid substitution, illustrating that a neurodegenerative process similar to a human disease can be genetically modeled in animals.     

人胰岛素原基因在转基因小鼠乳汁中的表达

将构建的绵羊 β-乳球蛋白基因 ( BLG)调控人胰岛素原基因的重组基因通过显微注射生产转基因小鼠。共移植 888枚注射 BLG-胰岛素原基因的小鼠卵 ,移植受体 31只 ,怀孕 1 4只 ,产仔 53只。PCR检测 51只 ,有 5只为阳性 ,并均为雌性 ,其中 2只小鼠泌乳 ,经放免检测小鼠乳汁中人胰岛素原的质量浓度分别为 37.44mg/L和 39.99mg/L。另外 3只异常消瘦而不孕 ,其中 1只极度衰竭而死亡     

A chicken transferrin gene in transgenic mice escapes X-chromosome inactivation

Mammalian X-chromosome inactivation involves a coordinate shutting down of physically linked genes. Several proposed models require the presence of specific sequences near genes to permit the spread of inactivation into these regions. If such models are correct, one might predict that heterologous genes transferred onto the X chromosome might lack the appropriate signal sequences and therefore escape inactivation. To determine whether a foreign gene inserted into the X chromosome is subject to inactivation, transgenic mice harboring 11 copies of the complete, 17-kilobase chicken transferrin gene on the X chromosome were used. Male mice hemizygous for this insert were bred with females bearing Searle's translocation, an X-chromosome rearrangement that is always active in heterozygous females (the unrearranged X chromosome is inactive). Female offspring bearing the Searle's translocation and the chicken transferrin gene had the same amount of chicken transferrin messenger RNA in liver as did transgenic male mice or transgenic female mice lacking the Searle's chromosome. This result shows that the inserted gene is not subject to X-chromosome inactivation and suggests that the inactivation process cannot spread over 187 kilobases of DNA in the absence of specific signal sequences required for inactivation.     

Inherited electrophoretic hemoglobin patterns among 20 inbred strains of mice

The hemoglobin from mice of six inbred strains is of the single-spot electrophoretic type, and that from 14 inbred strains is of the diffuse type. No selective advantage is apparent for either type. The distribution among strains shows some relation to the history of the development of the strains.     

Gonadotroph-specific expression of metallothionein fusion genes in pituitaries of transgenic mice

Transgenic mice expressing a metallothionein-somatostatin fusion gene contain high concentrations of somatostatin in the anterior pituitary gland, a tissue that does not normally produce somatostatin. Immunoreactive somatostatin within the anterior pituitaries was found exclusively within gonadotrophs. Similarly, a metallothionein-human growth-hormone fusion gene was also expressed selectively in gonadotrophs. It is proposed that sequences common to the two fusion genes are responsible for the gonadotroph-specific expression.     

Enhanced neurofibrillary degeneration in transgenic mice expressing mutant tau and APP

JNPL3 transgenic mice expressing a mutant tau protein, which develop neurofibrillary tangles and progressive motor disturbance, were crossed with Tg2576 transgenic mice expressing mutant beta-amyloid precursor protein (APP), thus modulating the APP-Abeta (beta-amyloid peptide) environment. The resulting double mutant (tau/APP) progeny and the Tg2576 parental strain developed Abeta deposits at the same age; however, relative to JNPL3 mice, the double mutants exhibited neurofibrillary tangle pathology that was substantially enhanced in the limbic system and olfactory cortex. These results indicate that either APP or Abeta influences the formation of neurofibrillary tangles. The interaction between Abeta and tau pathologies in these mice supports the hypothesis that a similar interaction occurs in Alzheimer's disease.