人促血小板生成素在转基因小鼠乳腺中定位表达的研究

通过转基因动物乳腺生物反应器大规模生产药用蛋白质已成为现代生物技术新的生长点之一。为研制表达人促血小板生成素的哺乳动物生物反应器的转基因小鼠模型,本论文以小鼠乳清酸蛋白（mWAP)基因5‘端调控区和牛α-sl-酪蛋白基因3‘端调控区作为调节元件构建了用于表达人促血小板生盛开纱的乳腺组织特异性表达载体pWAPTPO(Fig.l)。通过常规显微注射的方法把mWAP启动子指导的hTPO表达载体导入小鼠受精卵,获得出生小鼠16只,经PCR检测,有6只为转基因阳性（Fig.2)。G0代小鼠中转基因整合率为37.5%(6/16),用ELISA方法在G0代转基因雌鼠的乳汁中检测了促血小板生成素的表达,表达量在0.8μg/mL以上（Tablel)。这些结果表明我们已建立了乳腺表达hTPO的转基因小鼠模型,为以后大型家畜乳腺生物反应器的研制提供了科学依据。     

转基因小鼠乳腺表达人胰岛素基因的研究

转基因动物乳腺生物反应器表达和生产医用蛋白是国际上的研究热点,目前已有很多成功的转基因动物乳腺表达出外源蛋白质［１］。在转基因动物乳腺表达的蛋白质包括凝血因子Ⅸ、组织纤溶酶原激活物（ｔＰＡ）、α１抗胰蛋白酶原、白介素２、蛋白质Ｃ、超氧化物歧化酶...     

转基因小鼠乳腺表达G-CSF的研究

用PCR法从正常中国人脐带血提取总DNA作为模板,扩增出1.5 kb的人G-CSF基因组基因。序列分析证实其正确性。将其插入小鼠乳清酸蛋白(WAP)基因的起始密码子ATG前的KpnⅠ位点,使其受控于2.6kb的WAP调控序列,构建成乳腺表达载体pWGG。回收经EcoRⅠ酶切后的8.7kb片段用于显微注射。共注射1200枚受精卵,移植34受体母鼠,产仔鼠85只。经PCR检测和DNA印迹分析,证实获得两只整合有人G-CSF基因的雄性鼠,整合率为2.37％。建立的转基因鼠系表明,采用ELASA方法对F1代雌鼠乳汁检测,成功地表达出人G-CSF。表达量为120～250ng/ml。这一结果表明转基因的表达具有乳腺特异性。这为在大动物中实施转基因提供了依据。     

SOEing法在构建人瘦蛋白乳腺表达载体中的应用

目的 :将奶牛CSN2 5′端启动子区与人瘦蛋白cDNA序列进行拼接 ,进而构建人瘦蛋白乳腺特异性表达载体。方法 :设计特殊的“巨型引物” ,运用PCR方法分别从质粒pBBC和pHL上扩增了牛CSN2 5′端启动子 ( 2 8kb)和有完整读码框的人瘦蛋白基因。利用改进的重叠区扩增基因拼接法 (SOEing法 ) ,将两个独立的片断进行了拼接。结果 :得到了两者线形融合基因 ,为构建人瘦蛋白乳腺特异性表达载体打下了基础。     

人α-乳清蛋白基因的克隆及其在转基因小鼠中高效表达

从粘粒文库中筛选出人α-乳清蛋白基因,构建9.5 kb的转基因表达载体.利用显微注射的方法获得68只F₀代小鼠,经PCR检测和DNA印迹分析证实有8只小鼠（4♂,4♀）为整合人α-乳清蛋白基因的转基因阳性小鼠,整合率为11.7%,整合拷贝数在1至8之间.利用SDS-聚丙烯酰胺凝胶电泳检测和蛋白质印迹分析,4只雌性F₀代转基因阳性小鼠全部表达了人α-乳清蛋白.放射免疫测定法测定,含量分别为0.62 g/L、0.48 g/L、0.56 g/L、3.21 g/L;同时测定F₀代50号转基因公鼠的后代阳性母鼠（50-2号）乳样中人α-乳清蛋白含量也达到1.03 g/L,证明由原代转基因公鼠遗传给后代的人α-乳清蛋白基因亦获得了稳定的表达.所构建的人α-乳清蛋白转基因载体具有结构较小,表达量高,可以稳定遗传等优点.为利用人α-乳清蛋白基因改善牛乳成分和品质奠定了基础.     

人G—CSF基因组基因的克隆及其在转基因小鼠乳腺表达的研究

采用ＰＣＲ方法以正常中国人脐带血提取总ＤＮＡ为模板,扩增出１．５Ｋｂ的粒细胞集落刺激因子（Ｇ－ＣＳＦ）基因组基因,序列分析证实其正确性,将其插入小鼠乳清酸蛋白（ＷＡＰ）基因的起支密码子ＡＴＧ臆的ＫｐｎＩ位点,使其受控于２．６ｋｂ的ＷＡＰ调控序列,从而构建乳腺表达载体ｐＷＧＧ。回收经ＥｃｏＲＩ酶切后的８．７ｋｂ片段用于显微注射,共注射１２００枚受精卵,移植至受体３４母鼠,产生仔鼠８５只,经ＰＣＲ检测     

人尿激酶原乳腺定位转基因小鼠的建立

应用大鼠β乳酪蛋白基因的上游调控序列和人尿激酶原ｃＤＮＡ构建成功了乳腺定位表达载体．用显微注射的手段导入到受精卵的雄前核,从注射的３００枚受精卵中,１４０枚被移植到９只假孕的受体小鼠．结果从获得的子一代小鼠中,经ＰＣＲ和Ｓｏｕｔｈｅｒｎｂｌｏｔ证实,有３只转基因阳性的小鼠．     

绿色荧光蛋白基因在转基因小鼠体内的表达

建立绿色荧光蛋白（GFP）转基因小鼠,继而传代建系。采用显微注射法,将GFP基因注入FVB／NJ小鼠受精卵原核内,获得子代鼠。分娩后3周剪取仔鼠尾,提取基因组DNA,应用PCR、Southern印迹技术进行整合检测。结共用雌性小鼠200只,注射受精卵1586枚,移植卵数386枚,受体鼠32只,怀孕鼠4只,子代鼠18只,有4只为阳性：取2只首建鼠的胚胎,在荧光显微镜下观察GFP表达明显,表明初步获得了转绿色荧光蛋白基因小鼠,     

丙型肝炎核心蛋白与NS3蛋白在转基因小鼠中的表达

为研究丙型肝炎病毒核心蛋白及ＮＳ３蛋白在转基因小鼠体内的基因表达及细胞内定位,以及病毒蛋白 直接致细胞病变效应,我们采用ＲＴ－ＰＣＲ方法检测了靶基因ｍＲＮＡ在转基因小鼠各种组织中的表达,用免疫组化方法分析了核心蛋白与ＮＳ３蛋白在转基因小鼠体内的表达及细胞内定位。     

转基因动物的乳腺表达

转基因动物乳腺组织特异性表达异源基因是近年来基因工程中引人注目的途径.文章介绍了这一途径有关的乳汁蛋白基因、乳汁蛋白基因与异源基因的融合方式、重组基因的必要构成以及可能影响高效表达的因素.     

组织纤溶酶原激活剂突变体(La-tPA)转基因鼠的建立

用羊β－乳球蛋白基因（ＢＬＧ）５区５×１０３ｂ（１０３ｂ,旧称ｋｂ）为调控序列,构建了乳腺表达组织纤溶酶原激活剂突变体（Ｌａ－ｔＰＡ）载体．对５４０枚小鼠受精卵进行显微注射,经ＰＣＲ和Ｓｏｕｔｈｅｒｎｂｌｏｔ检测,获得６只整合有人Ｌａ－ｔＰＡ的转基因小鼠,整合率为３２％．这为未来利用转基因动物生产Ｌａ－ｔＰＡ提供依据     

人促血小板生成素在转基因小鼠乳腺中定位表达的研究(英文)

通过转基因动物乳腺生物反应器大规模生产药用蛋白质已成为现代生物技术新的生长点之一。为研制表达人促血小板生成素的哺乳动物生物反应器的转基因小鼠模型,本论文以小鼠乳清酸蛋白 (mWAP) 基因5挾说骺厍团-s1-酪蛋白基因3挾说骺厍魑鹘谠菇擞糜诒泶锶舜傺“迳伤氐娜橄僮橹匾煨员泶镌靥錺WAPTPO(Fig.1)。通过常规显微注射的方法把mWAP启动子指导的hTPO表达载体导入小鼠受精卵,获得出生小鼠16只。经PCR检测,有6只为转基因阳性(Fig.2)。G0代小鼠中转基因整合率为37.5% (6/16),用ELISA方法在G0代转基因雌鼠的乳汁中检测了促血小板生成素的表达,表达量在0.8 mg/mL以上(Table 1)。这些结果表明我们已建立了乳腺表达hTPO 的转基因小鼠模型,为以后大型家畜乳腺生物反应器的研制提供了科学依据。     

Expression of a Truncated Brca1 Protein Delays Lactational Mammary Development in Transgenic Mice

To address the hypothesis that certain disease-associated mutants of the breast-ovarian cancer susceptibility gene BRCA1 have biological activity in vivo, we have expressed a truncated Brca1 protein (trBrca1) in cell-lines and in the mammary gland of transgenic mice. Immunofluorescent analysis of transfected cell-lines indicates that trBRCA1 is a stable protein and that it is localized in the cell cytoplasm. Functional analysis of these cell-lines indicates that expression of trBRCA1 confers an increased radiosensitivity phenotype on mammary epithelial cells, consistent with abrogation of the BRCA1 pathway. MMTV-trBrca1 transgenic mice from two independent lines displayed a delay in lactational mammary gland development, as demonstrated by altered histological profiles of lobuloalveolar structures. Cellular and molecular analyses indicate that this phenotype results from a defect in differentiation, rather than altered rates of proliferation or apoptosis. The results presented in this paper are consistent with trBrca1 possessing dominant-negative activity and playing an important role in regulating normal mammary development. They may also have implications for germline carriers of BRCA1 mutations.     

Targeted Expression of the only Zinc Finger Gene in Transgenic Mice is Associated with Impaired Mammary Development

The only zinc finger (OZF) gene encodes a protein consisting mainly of 10 zinc finger motifs of the Krüppel type of yet unknown function. To potentially assess its in vivo role, mammary targeted deregulation of the expression of the murine gene was performed in transgenic mice using a goat -casein-based transgene. Mammary expression of the transgene was observed in the 11 lines obtained. In three expressing lines, this expression was tissue-specific and developmentally regulated. Further analysis of mice from two expressing lines revealed that transgene-homozygous females could not sustain full growth of their pups. This phenotype was associated with an impaired mammary gland development noticeable only after mid-gestation. It was characterised by an increase of the adipocyte to acini ratio and low or absence of fat globules within these acini compared to non-transgenic control animals. These transgenic observations strongly suggest that OZF is active in the mammary gland, interfering with the lactation process and thus that the described transgenic mice could be useful models to search for the cellular partner(s) of this protein.     

Dynamic Control of Oligosaccharide Modification in the Mammary Gland: Linking Recombinant Human Erythropoietin

We analyzed two transgenic mouse lines that secrete rhEPO in their milk to assess the dynamic control of N-linked oligosaccharides. Since pharmaceutically available epoetin α and β are produced in CHO cells, we compared transgenic mammary gland-derived rhEPO to its CHO cell-derived counterpart. The major glycosyltransferases that determine the N-oligosaccharides patterns of rhEPO include N-acetylglycosaminyltransferase (GnT) and α1,3/4 fucosyltransferase (Fuc-TIV), GnT-III, -V and Fuc-TIV expression in the mouse mammary gland is significantly higher than that in Chinese hamster ovary (CHO)-derived cells, where the protein is not detectable. The data suggest that N-linked sugar chain patterns of recombinant glycoproteins, produced by the mammary gland differ, since GnT-III alters the sugar pattern extensively. In our experiments, rhEPO produced by the transgenic mice contains more tetra-acidic oligosaccharide structures than epoetin α derived from CHO cells, a rhEPO that is widely used therapeutically. Accordingly, we examined milk-derived rhEPO activity, both in vitro and in vivo. The rhEPO protein purified from the milk of mammary glands upregulates the EPO receptor-mediated expression of the STAT5 gene in MCF-7 cells in a dose-dependent manner, similar to the effects of epoetin α. Furthermore, direct injection of rhEPO into the mouse tail vein leads to an increase in the levels of blood components, such as red blood cells and platelets. In light of these findings, we suggest that the mammary glands of transgenic animals provide a sufficient environment to generate rhEPO with post-translational modifications for biopharmaceutical use. These authors are equal contributors to this work.     

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。     

人肾素转基因小鼠的建立

为研究人肾素基因在体内的功能和建立其药物干预实验的动物模型,采用显微注射法,将纯化的人肾素基因导入小鼠受精卵,再培育成转基因小鼠.通过DIG DNA印迹和PCR分析,进行转基因整合检测.在出生的13只子代鼠中,得到一只转基因阳性鼠.整合率为7.7%,有效率0.3%,转基因已稳定传代.RT-PCR显示转基因阳性鼠的肾、心和肺组织中有肾素基因表达,而在肝脏与骨骼肌中则未检测到.阳性鼠血浆肾素活性较对照鼠明显升高,而肾与心脏组织的肾素活性则无明显变化.人肾素转基因小鼠可用于研究循环或组织的RAS中肾素基因的功能及有关其药物抑制实验.