转hDAF基因小鼠的构建(英文)

本工作构建了含有ｈＤＡＦ基因的转基因小鼠,以便研究ｈＤＡＦ基因能否消除异种器官移植中的排斥反应。 采用ＤＮＡ重组的方法构建ｈＤＡＦ基因的表达载体ｐＳＰ６４ＨＰ（Ｆｉｇ．１）。通过受精卵显微注射,将其中的目的基因片段,转移到小鼠体内,建立转基因小鼠。再通过Ｄｏｔ ｂｌｏｔｔｉｎｇ和Ｓｏｕｔｈｅｒｎ ｂｌｏｔｔｉｎｇ杂交方法对出生小鼠的基因组特征进行查证。 连续两次对直接裂解菌液做ＰＣＲ扩增,筛选出重组质粒（Ｆｉｇ．２＆３）,酶切图谱（Ｆｉｇ．４）和Ｓｏｕｔｈｅｒｎ杂交（Ｆｉｇ．５）分析结果与预期吻合,出现预期条带;小鼠受精卵注射后存活比率为７７．９％,受精卵的发育率为３．４％,出生小鼠中,１０．５％出现清晰杂交信号。 表明：ｈＤＡＦ基因表达载体构建成功;并整合入小鼠基因组中。     

Establishment of La-tPA/G-CSF dual transgenic mice and expression in their mammary gland

Expression vectors of human granulocyte colony stimulating factor (G-CSG) and long acting tissue plasminogen activator (La-tPA) in mammary gland were constructed using promoters of mouse whey acid protein gene (WAP) and sheep β-lactoglobulin gene (BLG) with sizes of 2.6 and 5 kb respectively. Two kinds of transgenic mice of G-CSF and La-tPA were produced with microinjection. The expression of G-CSF and La-tPA was achieved in mammary glands of transgenic mice, respectively. In order to establish dual transgenic mice of La-tPA/G-CSF, transgenic mice carrying G-CSF and La-tPA gene characterized with specific expression in mammary gland were mated. La-tPA/G-CSF dual transgenic mice were screened out from the hybrid offspring by Once-PCR. The co-expression of La-tPA and G-CSF in mammary gland of the dual transgenic mice was confirmed by the milk assayed and Northern blot analysis. Some parameters about the dual transgenic mice indicated that there were fewer litters than that of normal mice. The ratio of du     

胰腺组织表达Cre重组酶转基因小鼠的建立及鉴定

组织特异性表达Cre重组酶的转基因小鼠是进行组织特异性基因剔除研究的重要工具。为了建立胰腺组织特异性Cre转基因小鼠,我们通过PCR克隆了大鼠胰岛素基因启动子,并用它指导Cre基因在胰岛细胞中的特异性表达。在Cre重组酶基因5′端添加了真核核糖体结合序列和核定位序列以使Cre重组酶能穿越核膜在细胞核中发挥功能;同时,在Cre基因3′端添加了含内含子的3′端人生长激素基因。表达载体经显微注射导入小鼠受精卵以建立转基因小鼠。PCR检测显示共获得7只Cre整合阳性的转基因首建者小鼠;RTPCR结果表明其中1只首建者小鼠的子代鼠在胰腺中转录了外源基因,进一步的Southern杂交结果表明,该转基因小鼠能够在胰腺中表达有功能的Cre重组酶。      

转基因动物研究新进展

我们曾对转基因动物的制作方法、转基因动物研究的应用、转基因的表达特征及提高转基因表达的策略等作过专题综述＾「１」。而这之后在转基因动物的研究方面又获得了许多新进展,其中转线粒体动物的问世拓宽了转基因动物的研究内容。在制作转基因动物的方法上,１９９８－１９９９年世界上先后报道了三种新方法：即由Ｓｃｈｎｉｅｋ等报道的体细胞核移植技术实现转基因,由Ａｎｔｈｏｎｙ Ｗ．Ｓ．Ｃ等报道的通过用逆转录病毒载体感     

Transgenesis in rats: Technical aspects and models

The production of transgenic rats by DNA-microinjection into fertilizer ova has now become an established procedure, although fewer than 20 lines have been described during the last 5 years. Overall, transgenic rats remain more difficult to produce than transgenic mice, but satisfactory yields have been obtained by several laboratories. A review of the methods used to generate transgenic rats shows considerable variation between different laboratories, particularly in choice of strain, superovulation protocols and the use of embryo culture before reimplantation. In some instances, the production of transgenic rats has provided data that are new and relevant, compared to data obtained in mice bearing the same transgene. Models have been developed for human diseases such as hypertension and autoimmunity, and applications have been found in the study of carcinogenesis and in pharmacological research. Transgenic rat technology also opens up interesting perspectives for transplantation research, in which microsurgery is an essential procedure. Intensive research is in progress in several laboratories to produce rat embryonic stem (ES) cell lines, but existing lines have not participated in germ line formation a prerequisite for their use in gene knock out experiments.     

外源性人TIMP-1基因在转基因小鼠染色体上的整合及定位

为探讨外源基因人基质金属蛋白酶组织抑制物-1（human tissue inhibitor of metalloproteinase-1, hTIMP-1）基因在转基因小鼠家系染色体上的整合和精确定位,应用Southrn印迹检测外源基因在染色体上整合的位点及拷贝数.结果表明,外源基因是以单拷贝、单位点形式整合;应用荧光原位杂交（fluorescence in situ hybridization, FISH）技术检测F4～F20代转基因小鼠中外源基因的整合.结果证明,该家系转基因小鼠自F4代起是纯合子,外源基因整合在17号染色体E区;反向PCR法（Inverse PCR, IPCR）克隆出约3.8 kb外源基因整合位点处的侧翼序列.分析表明,外源基因整合在17号染色体E1.3区,ALK（anaplastic lymphoma kinase, ALK）基因第23个内含子区域.结果提示,获得的转基因小鼠为纯系,外源基因hTIMP-1已稳定整合在转基因小鼠染色体上,并能遗传给后代.     

Lactotroph hyperplasia in the pituitaries of female mice expressing high levels of bovine growth hormone

PEPCK/bGH transgenic mice have very high blood levels of foreign GH, and prominent reproductive disturbances, especially in females. To obtain a deeper insight into the causes of these abnormalities, pituitaries of PEPCK/bGH transgenics were studied by immunocytochemistry, electron microscopy and in situ hybridization (ISH) techniques. Pituitary weights were significantly reduced (P < 0.05) in transgenic males, while in transgenic females they were increased without reaching significance compared to nontransgenic controls. In both sexes, GH cells were inhibited, as previously described in other lines of GH transgenic mice. In females, PRL cells were increased by 37% compared to controls. Ultrastructurally, the lactotrophs had characteristics of stimulation and PRL mRNA was increased by 35%. In males the increase in the number of PRL immunoreactive cells was not significant, the PRL mRNA signal did not differ from controls, and there were no changes in their ultrastructure. Only in females ACTH cells were significantly reduced (P < 0.05) in number and unchanged in males; however, POMC mRNA signal was increased in both genders and reached significance (P < 0.05) in males. In females, but not in males, the percentage of LH cells was lower than in control mice. In conclusion, the high blood bGH levels induced sex related changes in transgenic mice from the present line. The infertility of PEPCK/bGH transgenic females may be attributed to lactotroph hyperplasia and marked reduction in number of gonadotrophs.     

人DAF基因在转基因小鼠中的遗传与表达

为研究人ＤＡＦ基因在小鼠体内遗传与表达的规律,从质粒ｐＳＦＦＶ－ＤＡＦ分离出一段包含人ＤＡＦ基因的ＤＮＡ片段。采用受精卵显微注射法建立转人ＤＡＦ基因小鼠。提取出生小鼠的染色体ＤＮＡ,经Ｄｏｔ－ｂｌｏｔ与Ｓｏｕｔｈｅｒｎ－ｂｌｏｔ杂交相结合确定首建转基因小鼠,并经Ｄｏｔ－ｂｌｏｔ杂交研究人ＤＡＦ基因在转基因小鼠体内的遗传特征,Ｎｏｒｔｈｅｒｎ杂交确定其表达情况。小鼠受精卵经基因导入后,共生出２４只小鼠,其中４只被确定为首建转基因小鼠,整合率为１５％,在首建转基因小鼠两两交配生出的Ｆ１代小鼠中分别有７０％和７５％继续携带人ＤＡＦ基因。首建转基因小鼠中有１只小鼠在ＲＮＡ水平表达了人ＤＡＦ基因。可见,人ＤＡＦ基因整合入小鼠基因组中,并能够稳定遗传及表达。     

角蛋白启动子在人乳头瘤病毒的转基因小鼠中的研究进展

人乳头瘤病毒（Human Papillomavirus,HPV）感染与多种疾病相关,但引起这些疾病的机制还不是很清楚。因此,人们想通过构建动物模型来深入了解HPV引起的各种疾病的机制。HPV具有严格的嗜人类组织的特性,这就要求在构建转基因动物模型中利用不同外源启动子,使HPV癌蛋白定向表达在特定的组织细胞中。本文主要介绍人角蛋白启动子在HPV的转基因小鼠中的一些研究进展。     

利用植物表达外源蛋白需解决的几个关键问题

植物外源蛋白表达系统已取得较大进展。目前已经能够利用转基因植物表达和生产多种外源蛋白,但仍存在诸多难以克服并急需解决的技术问题。作者从植物组织再生率、转化频率、调控元件、转基因植物遗传稳定性及其潜在的安全性问题、外源蛋白在植物体内的表达、调控、稳定性及其下游加工问题等方面综述了植物外源蛋白表达系统存在的问题和应对策略,为进一步开发、利用转基因植物生产外源蛋白提供理论依据。     

性别及日龄对转人神经生长因子基因小鼠唾液中蛋白分泌的影响

神经生长因子(Nerve growth factor,NGF)是一种能促进神经元发育、分化、再生的蛋白。为高效生产药效更佳的人源NGF (hNGF)药物,最近,笔者实验室构建出唾液腺特异表达hNGF的转基因小鼠,并从该转基因小鼠唾液中纯化获得具有高生物学活性的h NGF蛋白。为了选择性别和日龄最适宜的转hNGF基因小鼠用于收集纯化hNGF蛋白,文中比较了28日龄(性成熟前)雄性、雌性,63日龄(性成熟后)雄性、雌性转hNGF基因小鼠,共4组转hNGF基因小鼠分泌的唾液量、唾液总蛋白量、唾液鼠源NGF (mNGF)蛋白量和唾液h NGF蛋白量等指标。结果显示,63日龄的转hNGF基因小鼠分泌的唾液量、唾液总蛋白量、唾液mNGF蛋白量和唾液hNGF蛋白量显著高于28日龄同一性别的转hNGF基因小鼠,且63日龄的雄性转hNGF基因小鼠分泌的唾液hNGF蛋白量显著高于同一日龄的雌性转hNGF基因小鼠;在4组小鼠中,63日龄的雄性转hNGF基因小鼠分泌的唾液hNGF含量最高,比28日龄雌性转hNGF基因小鼠高出约46倍,最适宜用于收集唾液并从中纯化hNGF。     

通过细胞内的靶向定位大幅度提高外源蛋白在转基因植株的积累水平

通过体外操作,对豇豆胰蛋白酶抑制剂(cpti)基因进行修饰,获得了一个融合蛋白基因(sck).该基因是在cpti基因的基础上,在其5'端添加了信号肽编码序列,在3'端添加了内质网滞留信号编码序列,旨在引导基因转译产物进入细胞内质网,并最终滞留在内质网及其衍生的蛋白体内.用sck基因转化烟草(Nicotiana tabacum L.),对获得的转基因植株进行ELISA检测.结果表明,含有修饰基因的转基因烟草CpTI蛋白含量有明显提高,比转未修饰cpti基因烟草平均高出2倍,最高单株可达4倍以上,同时转基因植株的抗虫性也有了显著的提高.结果表明,采用外源蛋白靶向定位的策略,可大幅度提高外源蛋白在转基因植物细胞内的积累量,在植物基因工程研究中具有广泛的借鉴意义.     

Smad6/Smurf1 overexpression in cartilage delays chondrocyte hypertrophy and causes dwarfism with osteopenia

Biochemical experiments have shown that Smad6 and Smad ubiquitin regulatory factor 1 (Smurf1) block the signal transduction of bone morphogenetic proteins (BMPs). However, their in vivo functions are largely unknown. Here, we generated transgenic mice overexpressing Smad6 in chondrocytes. Smad6 transgenic mice showed postnatal dwarfism with osteopenia and inhibition of Smad1/5/8 phosphorylation in chondrocytes. Endochondral ossification during development in these mice was associated with almost normal chondrocyte proliferation, significantly delayed chondrocyte hypertrophy, and thin trabecular bone. The reduced population of hypertrophic chondrocytes after birth seemed to be related to impaired bone growth and formation. Organ culture of cartilage rudiments showed that chondrocyte hypertrophy induced by BMP2 was inhibited in cartilage prepared from Smad6 transgenic mice. We then generated transgenic mice overexpressing Smurf1 in chondrocytes. Abnormalities were undetectable in Smurf1 transgenic mice. Mating Smad6 and Smurf1 transgenic mice produced double-transgenic pups with more delayed endochondral ossification than Smad6 transgenic mice. These results provided evidence that Smurf1 supports Smad6 function in vivo.     

转基因动物研究进展及前景

本文综述了国内外转基因动物的概念、制备、意义、应用、特点等方面的研究进展,系统分析了转基因研究中的问题及其原因,展望了转基因动物的前景。     

Ubiquitous expression of mRFP1 in transgenic mice

Fluorescent proteins provide a powerful means to track gene expression and cellular behaviors in the study of model organisms such as mice. Among the new generation of fluorescent protein markers, the monomeric red fluorescent protein mRFP1 is particularly attractive because of its rapid maturation and minimal interference with GFP and GFP-derived markers. Here we evaluate the utility of mRFP1 as a marker in transgenic mice. We show that high level and ubiquitous expression of mRFP1 does not affect mouse development, general physiology, or reproduction. mRFP1 expression can be readily detected with unaided eyes under daylight in transgenic mice on the albino background. The intensity of mRFP1 signals can be used to distinguish homozygous and heterozygous transgenic mice. Together, these features make mRFP1 an attractive marker for broad applications in transgenic research.     

Transgenic mice with pancellular enhanced green fluorescent protein expression in primitive hematopoietic cells and all blood cell progeny

Transgenic mice homogeneously expressing enhanced green fluorescence protein (EGFP) in primitive hematopoietic cells and all blood cell progeny, including erythrocytes and platelets, have not been reported. Given previous data indicating H2Kb promoter activity in murine hematopoietic stem cells (HSCs), bone marrow (BM), and lymphocytes, an H2Kb enhancer/promoter EGFP construct was used to generate transgenic mice. These mice demonstrated pancellular EGFP expression in both primitive BM Sca-1+Lin-Kit+ cells and side population (SP) cells. Additionally, all peripheral blood leukocytes subsets, erythrocytes, and platelets uniformly expressed EGFP strongly. Competitive BM transplantation assays established that transgenic H2Kb-EGFP HSCs had activity equivalent to wildtype HSCs in their ability to reconstitute hematopoiesis in lethally irradiated mice. In addition, immunohistochemistry revealed EGFP transgene expression in all tissues examined. This transgenic strain should be a useful reagent for both murine hematopoiesis studies and functional studies of specific cell types from particular tissues.     

Early Changes in Behavior Deficits, Amyloid β-42 Deposits and MAPK Activation in Doubly Transgenic Mice Co-expressing NSE-Controlled Human Mutant PS2 and APPsw

1. Doubly transgenic mice were some differences in the period proceeding of the development of Abeta-42 deposits and behavioral deficits. It was not characterized human mutant PS2 (hPS2) with APPsw in the brains of double transgenic mice. The aim of this study was to examine whether doubly transgenic mice co-expressing NSE-controlled APPsw and hPS2m develop AD-like phenotypes much earlier than singly APPsw or hPS2m alone. 2. We produced doubly transgenic mice from a cross between our previously created NSE-controlled hPS2m and an APPsw transgenic line. This doubly transgenic line was quantitatively produced by cross with age-matched control mice, and the produced mice were separated into 5, 6, 7 and 8-month old age groups. At the age of 8 months, the four groups of mice were tested for behavioral function, levels of Abeta-42 deposition, and potential signaling events. 3. It was shown that all the AD-like phenotypes, including behavior deficits, Abeta-42 levels, MAPK activation and ER expressions in doubly transgenic mice develop much earlier in the early time of AD development than their singly transgenic and non-transgenic littermates. 4. The results suggest that elevated Abeta-42 levels, and MAPK activation in doubly transgenic mice are model for early diagnosis and treatment of AD with therapeutic drug.     

Quantitative collection of milk and active recombinant proteins from the mammary glands of transgenic mice

Summary

Transgenic mice expressing foreign genes specifically in their mammary glands have been obtained by several groups in the world. The mouse is generally considered as a good reference animal to evaluate the efficiency of gene constructs to be used in larger mammals for the preparation of the corresponding recombinant proteins at an industrial scale. The method described here shows that mammary glands from lactating mice separated from their pups for one day spontaneously released 1.5 ml milk when stored at O'C. The proteins of milk obtained by this method were essentially similar to those obtained after milking. Human growth hormone (hGH) gene under the control of the rabbit whey acidic (WAP) gene promoter was expressed at a high level in the milk of transgenic mice (4 mg/ml milk in the mice examined here). hGH was present in milk obtained after milking or after the incubation of the mammary glands at O'C. In both cases, the hormone was present in essentially similar concentration, undegraded and biologically active (as judged by its prolactin‐like activity). The method depicted here is very simple and can be applied easily to many mice. Its major limitation is that it implies the breeding and the sacrifice of a relatively large number of animals. One gram of crude recombinant protein can be virtually obtained in this way with about 200 lactating mice from their milk containing the proteins at the concentration of 3‐4 mg/ml. The milk of transgenic mice can therefore be considered as a practical source of recombinant proteins for biochemical and pharmaceutical studies.     

Development of Transgenic Mice Expressing Calcitonin as a Beta-lactoglobulin Fusion Protein in Mammary Gland

Expression of foreign proteins in mammalian milk is becoming a widespread strategy for high-level production of recombinant pharmaceuticals, especially those with the most complex post-translational modifications. A milk-specific ovine beta-lactoglobulin (oBLG) promoter was used to drive expression of recombinant calcitonin in mouse milk. A gene construct was generated, consisting of 10.7 kbp of the oBLG gene including its promoter and 3′ flanking region with the calcitonin coding sequences inserted in-frame into the oBLG fifth exon. After microinjection, six founder mice transmitted the transgene to their progeny. RT-PCR confirmed mammary-gland specific expression of recombinant mRNA in most transgenic mice and Western blot analysis confirmed expression of chimeric protein. Calcitonin can thus be expressed under the oBLG promoter and regulatory elements in a mammary-gland specific manner.     

提高转基因表达水平的策略

随着转基因相关技术的发展,转基因动物技术在许多方面得到了成功应用.但外源基因在体内的表达仍然难以预测,特别是大动物的转基因,由于制备效率低下,因而难以筛选出足够的高表达的阳性动物数.基因表达调控研究对提高外源基因在动物体内的表达水平提供了一些新手段,本就避免转基因的位置效应、控制外源基因在动物宿主基因组中的整合、提高转基因的表达效率、构建转基因载体和使用外源基因需要注意的问题等进行综述.