牛β-乳球蛋白基因调控序列指导组织型纤溶酶原激活剂在小鼠乳腺中的表达

用全长8.4kb的牛β-乳球蛋白基因(BLG)作为调控序列,用1.6kb的鸡溶菌酶MAR序列作为对抗转基因中位点效应的工具,构建了组织型纤溶酶原激活剂(tPA)乳腺表达载体。对2300枚卵进行显微注射,经PCR和Southern\|Blot检测,在170只出生小鼠中获得9只整合有牛BLG-tPA融合基因的转基因小鼠,并在转基因小鼠乳汁中检测到tPA的活性,tPA的表达水平最高达到12μg/mL。整合在小鼠基因组中的牛BLGtPA融合基因能稳定地遗传给子代。     

转基因小鼠乳腺表达长效组织纤溶酶原激活剂

组织纤溶酶原激活剂(tPA)是一种较理想的溶血栓药物,本研究采用其突变体长效组织纤溶酶原激活剂(LAtPA)的cDNA作为目的基因,将其插入羊β-乳球蛋白(BLG)基因起始密码之前,使LAt-PA的转录、翻译受控于BLG基因的5′、3′序列,将此构建BLG-LAtPA用显微注射方法建立转基因鼠,经点杂交筛选和DNA印迹鉴定,获得2只LAtPA基因整合阳性鼠,并在阳性母鼠乳汁检测到有溶纤活性的LAtPA表达,表达水平1.5μg/ml。在这两只转基因鼠的F1代子鼠中,9只中有5只是阳性的,tPA表达水平维持在1-2μg/ml,说明BLG-tPA融合基因整合到小鼠基因组,能稳定地遗传给子代。为今后利用牛、羊作为生物反应器表达LAtPA奠定了基础。     

组织纤溶酶原激活剂突变体在转基因小鼠乳腺中的表达

组织纤溶酶原激活剂(tPA)是一种较理想的溶血栓药物,本研究采用其突变体——长效组织纤溶酶原激活剂(LAtPA)的cDNA作为目的基因,将它插入羊β-乳球蛋白(BLG)基因起始密码之前,使LAtPA的转录、翻译受控于BLG基因的5′、3′序列,再将所构建的BLG-LAtPA用显微注射方法建立转基因鼠,经点杂交筛选和Southern印迹鉴定,获得2只LAtPA基因整合阳性鼠,并在阳性母鼠乳汁中检测到有溶纤活性的LAtPA表达,表达水平1.5μg/ml。在这两只转基因鼠的9只F1代子鼠中,有5只是阳性的,tPA表达水平维持在1～2μg/ml,BLGtPA融合基因整合到小鼠基因组,能稳定地遗传给子代。     

乳腺注射法表达人组织型纤溶酶原激活剂的研究

目的 :构建tPA乳腺定位表达载体 ,使其在牛乳汁中高效表达 ,观察目的基因表达的规律及其影响因素 ,为建立新型牛乳腺生物反应器提供理论基础。方法 :RT-PCR法克隆目的基因 ,通过酶切、连接、分离、纯化等方法构建含tPA-cDNA的乳腺定位表达载体 ;采用乳腺注射法将融合基因转入小鼠及牛的乳腺组织中。结果 :乳腺注射外源基因后 ,tPA可在小鼠和牛的乳汁中表达。结论 :乳腺注射法可使目的基因在乳腺组织中稳定地表达较长的时间 ,其表达量与显微注射法没有明显的差异 ,表明外源基因的表达不受转基因方法的影响。但tPA在牛乳汁中的表达量明显高于小鼠的表达量 ,提示不同动物的乳蛋白调控系统有一定的差异 ,可能受着不同的因素或调控系统的影响。     

牛α—sl酪蛋白基因序列指导htPA突变体小基因在小鼠乳腺中的表达

将包括α－sl酪蛋白基因的启动子、LAtPA小基因、α－sl酪蛋白基因下游调控序列的融合基因经显微注射至小鼠的受精卵中,获得了5只转基因阳性鼠,其中1只转基因阳性雌鼠乳清中LAtPA的含量为0．18αg/ml,说明构建的LAtPA小基因能够正确表达出有生物活性的LAtPA, 在酪蛋白基因调控序列的指导下在小鼠乳汁中表达。     

组织纤溶酶活剂突变体在转基因小鼠乳腺中的表达

组织纤溶酶原激活剂（ｔＰＡ）是一种较理想的溶血栓药物,本研究采用其突变体－长效组织纤溶酶原激活剂（ＬＡｔＰＡ）的ｃＤＮＡ作为目的基因,将它插入羊β－乳球蛋白（ＢＬＧ）基因起始密码之前,使ＬＡｔＰＡ的转录、翻译受控于ＢＬＧ基因的５＇、３＇序列,再将所构建的ＢＬＧ－ＬＡｔＰＡ用显微注射方法建立转基因鼠,经点杂交筛选和Ｓｏｕｔｈｅｒｎ印迹鉴定,获得２只ＬＡｔＰＡ基因整合阳性鼠,并在阳性母鼠乳汁中检测到有     

ＢＬＧ—ＬＡtPA和鼠ＷＡＰ共注射提高ＬＡtPA在转基因小鼠乳腺中的表达水平

为了提高长效组织纤溶酶原活剂（ＬＡtPA）在转基因小鼠乳腺中的表达水平,将受控于羊β－乳球蛋白（ＢＬＧ）的ＬＡtPA表达载体ＢＬＧ－ＬＡtPA与小鼠乳清酸蛋白（ＷＡＰ）基因片段进行共注射,采用此方法建立的转基因小鼠,经ＰＣＲ筛选和Ｓouthern印迹鉴定,获得３只ＬＡtPA和ＷＡＰ共整合阳性鼠,并在阳性母鼠乳汁中检测到有溶纤活性的ＬＡtPA表达,表达水平达到１０μg/mL。     

组织型纤溶酶原激活剂的纯化制备

简述了用于大规模生产组织型纤溶酶原激活剂(tPA)的重组动物细胞及其培养工艺。从重组tPA的大规模、快速纯化的角度考虑,对tPA的纯化制备方法进行了简要评述。     

牛α-sl酪蛋白基因序列指导htPA突变体小基因在小鼠乳腺中的表达

将包括α－ｓｌ酪蛋白基因的启动子、ＬＡｔＰＡ小基因、α－ｓｌ酪蛋白基因下游调控序列的融合基 因经显微注射至小鼠的受精卵中,获得了５只转基因阳性鼠,其中１只转基因阳性雌鼠乳清中 ＬＡｔＰＡ的含量为０．１８μｇ／ｍｌ,说明构建的ＬＡｔＰＡ小基因能够正确表达出有生物活性的ＬＡｔＰＡ, 并且可在酪蛋白基因调控序列的指导下在小鼠的乳汁中表达。     

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     

RT-PCR测定组织纤溶酶原激活剂突变体(La-tPA)在转基因鼠乳腺表达的研究

利用所构建的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ＰＣＰ和Ｓｏｕｔｈｅｒｎｂｌｏｔ检测,获得６只整合有人Ｌａ－ｔＰＡ转基因小鼠．为了精确研究转基因在小鼠体内的表达,采用ＲＴ－ＰＣＰ方法测定转基因鼠在泌乳期１～２０ｄＬａ－ｔＰＡ基因的转录,结果表明,在转基因鼠乳腺的Ｌａ－ｔＰＡｍＲＮＡ水平在１０～１５ｄ最高,在２０ｄ时降为最低．外源基因在转基因小鼠乳腺的表达规律研究为未来利用转基因动物生产Ｌａ－ｔＰＡ提供依据     

Efficient BLG-Cre Mediated Gene Deletion in the Mammary Gland

Using the phage P1-derived Cre/loxP recombination system, we have developed a strategy for efficient mammary tissue specific inactivation of floxed genes. Transgenic mice were generated which express Cre DNA-recombinase under the control of the mammary gland specific promoter of the ovine beta- lactoglobulin (BLG) gene. To test the specificity of Cre mediated recombination, we crossed these mice to animals harbouring a floxed DNA ligase I allele. We show that the BLG-Cre construct specifies mammary specific gene deletion, and furthermore that it is temporally regulated, predominantly occurring during lactation. We fully characterised the extent of gene deletion in one line (line 74). In this strain the virgin gland is characterised by low levels (7%) of Cre mediated deletion, whereas 70–80% of cells within the lactating mammary gland have undergone recombination. Immunohisto-chemistry and indirect in situ PCR were used respectively to demonstrate that both Cre protein and Cre activity were evenly distributed throughout the population of secretory epithelial cells. The level of background recombination in non-mammary tissues was found to be 1.1%, irrespective of mammary gland developmental status. Crossing the transgenic BLG-Cre strain described here to mice harbouring other floxed alleles will facilitate the functional analysis of those genes during differentiation and development of the mammary gland.     

组织特异RNAi转基因小鼠模型的构建

RNAi是一种行之有效的基因沉默的新方法,被广泛地应用于基因功能的研究、疾病的治疗以及新型疫苗的研制等领域.本研究通过原核显微注射干扰载体的方法制备转基因小鼠.选用皮肤组织特异表达的人源角蛋白14（K14）基因启动子（2000bp）作为表达载体启动子,成功地驱动融合表达载体EGFP-shRNA进行干扰片段前体的转录,进而生成成熟的干扰片段,靶向小鼠BMP4基因使其发生沉默.所得到的转基因小鼠及其杂交后代经PCR和Southern杂交鉴定,结果表明外源基因准确无误地整合到小鼠基因组.Northern杂交结果证明,小干扰RNA在皮肤组织中有较高水平的表达,在肺和肠组织中有较低水平的表达.研究结果表明,利用PolⅡ型（K14）启动子驱动shRNA融合转录本的表达,在特定组织高表达siRNA,从而达到抑制特定组织目的基因表达的技术路线是可行的.同时为利用K14启动子进行毛囊相关基因干扰研究积累了基础数据,为制备组织特异抑制基因表达的转基因大家畜提供了一个参考方法.     

Transgenic Mice Expressing Recombinant Human Protein C Exhibit Defects in Lactation and Impaired Mammary Gland Development

To determine if the production of recombinant human protein C (rHPC) could be increased in milk, we created two lines of mice homozygous for the mouse whey acidic protein (WAP)/human protein C (HPC) transgene. Females of both lines had normal growth, activity and fertility, but failed to lactate normally and were unable to raise litters. Histological analyses of mammary glands from lactating homozygous females showed barely distended alveoli filled with dense-staining milk. Epithelial cells within these alveoli had distinct, centrally located nuclei and contained intracellular lipid droplets. Hemizygous animals derived from these lines were able to lactate and raised normal sized litters. Northern blot analysis showed that the 6.4 homozygous (6.4H) line expressed the transgene at higher levels then corresponding hemizygous (6.4) animals, but the 4.2 homozygous (4.2H) line expressed the transgene at lower levels than the 4.2 hemizygous line. The 6.4H line also had increased rHPC levels in the milk as revealed by western blot analysis. The 4.2H, 6.4, and 6.4H lines showed decreased and/or delayed expression of WAP, -casein, and -lactalbumin mRNA's compared to wild type animals during lactogenesis. The 4.2 line showed decreased mRNA expression for -casein and -lactalbumin, but normal or higher expression of WAP during lactogenesis. Elevated levels of some proteins were detected in the milk of transgenic mice. From these results, it is concluded that expression of rHPC induced a lactational phenotype that involves abnormal morphological, biochemical, and functional differentiation of mammary epithelial cells. However, the induction of this phenotype does not appear to be directly related to the level of rHPC mRNA expression, thus suggesting that the basis of this phenotype may involve secondary, rather than primary, effects of rHPC on mammary gland development.Deceased.     

用酵母α因子启动子前导肽系统表达tPA

本文首次选用酵母α-因子启动子前导肽系统有效地表达了组织型纤溶酶原激活剂基因。我们的实验结果表明3′端不翻译区的长度对表达有影响。     

血管内皮细胞特异表达Cre重组酶转基因小鼠的建立

血管内皮细胞参与血管形成、血管稳态维持、血栓形成、炎症和血管重建等生理和病理过程。为了便于通过Cre-LoxP系统研究相关基因在血管内皮细胞中的功能,创建了Tie2-Cre转基因小鼠,利用Tie2基因的启动子驱动Cre重组酶基因在血管内皮细胞中表达。经基因组PCR和Southern Blot鉴定有6只小鼠在基因组上整合有Cre基因,整合率为11％。为了验证Cre重组酶的剪切活性和表达组织分布,我们将Tie2-Cre转基因小鼠分别与Smad4条件基因打靶小鼠和报告小鼠ROSA26交配。Tie2-Cre;Smad4^co/＋小鼠的多个组织的基因组DNA的PCR结果显示,Cre重组酶在所有包含血管内皮细胞的组织中表达并能介导LoxP间的重组。Tie2-Cre;ROSA26双转基因胚胎LacZ染色结果显示,Cre重组酶在所有被检测组织的血管内皮细胞中特异性表达。因此．Tie2-Cre转基因小鼠可作血管内皮细胞谱系分析和在血管内皮细胞进行条件基因打靶的理想工具小鼠。     

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.     

转基因小鼠乳腺表达人瘦蛋白的研究

利用转基因动物乳腺生产药用蛋白质是近年来研究的热点,在这方面已有不少成功的例子,展现出良好的应用前景[1,2].本研究选择人瘦蛋白基因作为目标基因是因为其表达产物瘦蛋白能对人体内脂肪的蓄积和能量消耗进行有效的反馈调控,美国科学家已将用E.coli表达的人瘦蛋白用于人肥胖症的治疗并取得了良好的治疗效果[3],但尚未见到利用转基因动物乳腺表达这种蛋白质的研究报道.     

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