Expression of recombinant anticoagulant hirudin in the differentiated cultures of the porcine mammary epithelial cell line SI-PMEC

To express recombinant proteins in the spontaneously immortalized porcine mammary epithelial cell line (SI-PMEC) currently established in our laboratory, a chemically synthesized DNA fragment encoding the anticoagulant hirudin was used to construct a mammalian expression vector under the control of the goat beta-casein regulatory sequence. The vector, named pGB562/Hi, was transfected into the SI-PMEC cells to yield pGB562/Hi/SI-PMEC. The pGB562/Hi/SI-PMEC cells expressed recombinant hirudin only when they were differentiated into functional structures by growth on a Matrigel-coated petri dish supplemented with the lactogenic hormone prolactin. The differentiated pGB562/Hi/SI-PMEC cells produced about 0.5-0.6microg of recombinant hirudin/mg of total cellular protein. These results suggest that the established SI-PMEC cells have pharmaceutical potential to inducibly express bioactive heterogeneous proteins.     

人乳铁蛋白cDNA 基因乳腺表达载体的构建与鉴定

为了构建人乳铁蛋白基因 (hLF) 的乳腺表达载体并验证其在乳腺细胞中的表达情况,本载体以山羊β-casein基因上游包括启动子、外显子1、内含子1、部分外显子2作为5′端调控序列,下游包括部分外显子7、内含子7、外显子8、内含子8、外显子9及3′部分基因组片段作为3′端调控序列,长度分别为6.2 kb和7.1 kb,将hLF基因 (目的基因) 和Neo基因 (筛选标记) 分别插入到5′端调控序列和3′端调控序列的下游,构建成pBC1-hLF-Neo载体,其全长为25.348 kb。为了检测该载体的生物学     

Plasticity of the response of fetal mouse fibroblast to lactation hormones

In the work described in this article, mouse fetal fibroblasts were treated with three lactation hormones (insulin, progesterone and oxytocin) and the cellular changes were analyzed by RT-PCR-Southern hybridization. A gene-expression pattern characteristic of mammary epithelioid cells was induced by the hormones, as indicated by expression of the marker genes alpha-casein and beta-casein. Two mammary epithelial cell-specific gene expression vectors were constructed with bovine alpha-s1-casein or ovine beta-casein gene promoters directing an EGFP reporter gene. Transient expression of the EGFP gene was observed in cells treated by the hormones but not in control cells. Cell morphology also changed after insulin and oxytocin treatments; the cells resembled epithelial cells rather than fibroblasts. Our results suggest that mouse fetus fibroblasts can be partially induced by lactation hormones to resemble mammary epithelial cells. This procedure might help to increase the efficiency of gene targeting in studies of mammary gland bioreactors.     

牛乳腺上皮细胞的分离培养及其生物学特性

采用胶原酶消化法和胰蛋白酶选择性消化法分离、培养和纯化牛乳腺上皮细胞。形态学观察表明,培养的细胞具有典型的上皮细胞形态特征;染色体分析结果表明,培养的细胞具有正常的染色体数目。通过荧光免疫细胞染色方法鉴定了培养的细胞表达上皮细胞特异的角蛋白5和8。该细胞在添加胰岛素、氢化可的松以及羊催乳素的无血清培养液中诱导培养时,用RT-PCR方法检测到了β-酪蛋白基因的转录。这些结果表明,分离培养的细胞是乳腺上皮细胞,这些细胞在诱导培养的条件下能够转录表达β-酪蛋白。     

ht-Pam基因在山羊β-酪蛋白基因座定位整合的研究

利用体细胞基因打靶与核移植技术制备动物乳腺生物反应器是当今转基因定位整合表达的一种新技术。分别克隆山羊的β-酪蛋白基因5′调控区的6.3kb片段,外显子7、外显子8和9三个基因片段,并与克隆的人tPA突变体cDNA一起构建了含有neo和tk正负筛选标记基因的β-酪蛋白基因打靶载体P^GBC4tPA,并验证了neo基因、tk基因以及Cre-LoxP系统的有效性。将线性化的P^GBC4tPA通过电转染整合到山羊胎儿成纤维细胞基因组中,利用G418和GANC进行抗性细胞克隆的药物筛选,初步获得抗性细胞克隆244个,PCR检测后获得阳性细胞克隆31个,其中初步验证2个细胞克隆转植基因整合位点重组后的基因序列正确,并且该细胞克隆能够有效扩增。这为下一步基因打靶体细胞核移植制备山羊乳腺生物反应器奠定了基础。     

Lipotropes alter casein gene expression in bovine mammary acinar culture

Lipotropes (methyl group containing nutrients, including methionine and choline, folic acid, and vitamin B(12)) are essential for cell proliferation and differentiation of mammary tissues. Lipotropes interact in the supply and regulation of intracellular methyl group pools, thereby affecting synthesis and methylation of DNA. To determine the effect of lipotropes on milk protein gene expression, acini isolated from mammary tissues of lactating cows were cultured in one of three treatment media: (1) control, (2) lipotrope deficient, and (3) lipotrope supplemented. beta-Casein mRNA was determined by Northern blotting, and milk protein secretion was measured by a pulse-chase method. The level of beta-casein mRNA was lower in cells grown in lipotrope-deficient medium than in cells grown in the lipotrope-supplemented and control media. Acinar cells cultured in lipotrope-deficient medium also had approximately threefold less milk protein secretion than that of cells in either control or lipotrope-supplemented media. Protein secretion did not differ in the control and lipotrope-supplemented groups. The present study indicates that lipotrope deficiency suppresses total protein secretion and beta-casein gene expression in bovine mammary alveolar epithelial cells in culture.     

An efficient gene transfer system for hematopoietic cell line using transient and stable vectors

The hematopoietic system represents an interesting model for gene transfer protocols. Here, we have evaluated the efficiency of a gene transfer system using the polycationic compound SuperFect (Qiagen) and the K562 hematopoietic cell line. Transient and stable vectors carrying the enhanced green fluorescent protein (EGFP) reporter gene were employed. The stable vector was constructed based on Epstein-Barr virus sequences such as EBV oriP (origin of replication) and EBNA (EBV nuclear antigen)-1, both for DNA replication. The transfection efficiency of the viable cells was estimated by flow cytometry at approximately 98% for transient and stable vectors. Transiently transfected cells presented optimal EGFP expression until day 2 when fluorescence started to decrease. In contrast, stable transfectants continuously expressed the marker gene product for 10 weeks in the presence of G418. Our results represent an efficient gene transfer method for K562 hematopoietic cells and may be used as an alternative approach for further gene transfer studies involving hematopoietic cells.     

表达绿色荧光蛋白的重组CVI988病毒的构建及特性

提取马立克氏病毒Ⅰ型疫苗毒株CVI988的总DNA为模板,利用PCR技术扩增出病毒生长非必需的US2基因并克隆入T—easy载体。将CMV启动子和增强子控制的含GFP基因表达盒克隆入US2基因中,成功构建了含GFP基因的转移质粒载体pGUS2GFP。用脂质体将其与CVI988株共转染CEF细胞,用96孔板稀释法得到纯化的表达绿色荧光蛋白的重组CVI988病毒株rCVIGFP,并分别测定其在体内和体外的生长情况。表达EGFP基因的重组病毒在细胞上生长曲线与亲本毒CVI988类似,体外实验表明,1日龄腹腔接种该重组毒后,可以从鸡体内分离到表达绿色荧光的病毒。     

高原鼠兔瘦素蛋白乳腺特异表达载体的构建及细胞表达

瘦素(Leptin)蛋白是调节机体能量代谢的关键因子之一。前期研究显示高原鼠兔Leptin蛋白发生了适应性进化。功能实验表明,在温暖或寒冷条件下高原鼠兔Leptin通过减少食物摄取和增加能量消耗调节能量平衡,显示了其调节适应性产热过程的潜力。本研究以高原鼠兔Leptin cDNA为模板扩增高原鼠兔obese (ob)基因编码区序列504 bp,改造并构建哺乳动物真核细胞乳腺特异表达载体pBC1-lep,同时通过组织块法原代培养建立奶山羊乳腺上皮细胞系,并通过pBC1-lep质粒脂质体法转染及转基因细胞的筛选,成功获得转染Leptin的阳性细胞。本研究为利用转基因动物实现奶山羊乳腺中特异表达高原鼠兔Leptin提供了一条可能的途径,完成了乳腺特异真核表达载体的构建。     

Analysis of tissue-specific expression of human type II collagen cDNA driven by different sizes of the upstream region of the beta-casein promoter

To investigate the ability of 1.8 kb or 3.1 kb bovine beta-casein promoter sequences for the expression regulation of transgene in vivo, transgenic mice were produced with human type II collagen gene fused to 1.8 kb and 3.1 kb of bovine beta-casein promoter by DNA microinjection. Five and three transgenic founder mice were produced using transgene constructs with 1.8 kb and 3.1 kb of bovine beta-casein promoters respectively. Founder mice were outbred with the wild type to produce F1 and F2 progenies. Total RNAs were extracted from four tissues (mammary gland, liver, kidney, and muscle) of female F1 transgenic mice of each transgenic line following parturition. RT-PCR and Northern blot analysis revealed that the expression level of transgene was variable among the transgenic lines, but transgenic mice containing 1.8 kb of promoter sequences exhibited more leaky expression of transgene in other tissues compared to those with 3.1 kb promoter. Moreover, Western blot analysis of transgenic mouse milk showed that human type II collagen proteins secreted into the milk of lactating transgenic mice contained 1.8 kb and 3.1 kb of bovine beta-casein promoter. These results suggest that promoter sequences of 3.1 kb bovine beta-casein gene can be used for induction of mammary gland-specific expression of transgenes in transgenic animals.     

山羊β-casein位点打靶载体在乳腺上皮细胞中的表达研究

以本地山羊基因组DNA为模板,通过长链PCR扩增出山羊β-casein上游包括启动子,外显子1及部分外显子2的6.1kb的调控序列及下游3.3kb的序列,将来自质粒pCDNA3的neo基因以及来自质粒pNEOZTK-2的tk基因,经克隆重组后构建了本地山羊乳腺特异性定点打靶载体,并在其中克隆人乳铁蛋白mini基因,采用脂质体法转染小鼠乳腺上皮癌化细胞系C127,以进行打靶载体的表达功能检测,双夹心ELISA测得诱导液中乳铁蛋白表达量为0.2μg/mL,Western-blot显示重组蛋白分子量比标准品略小,约为76kD,结果说明本载体能够指导外源基因在动物乳腺细胞内正确表达。     

乳腺生物反应器特异高效表达载体的构建

乳腺生物反应器具有广阔的开发前景,而提高目的基因的表达量是该领域一个重要的研究课题。因此,选用强的非特异性启动子pCAG,而不是乳腺特异性启动子来实现高效表达;通过Cre/loxP系统和山羊β-乳球蛋白启动区（pBLG）表达Cre重组酶来实现载体的自删除以达到乳腺特异表达的目的。方法：构建含PolyA终止信号的Cre乳腺特异表达元件PolyA-pBLG-cre,插至强启动子pCAG驱动的报告基因lacZ表达载体中,构建成乳腺特异表达载体pCBCZ（pCAG-loxP-PolyA-pBLG-cre-loxP-lacZ）。转染细胞实验结果：PCR鉴定确认pCBCZ载体在小鼠乳腺上皮细胞（HC11）中发生Cre-loxP同源重组。X-Gal染色表明载体能驱动lacZ在HC11细胞中高效表达β-半乳糖苷酶,而在NIH 3T3细胞中仅少量表达。结论：构建的pCBCZ载体能高效驱动外源基因在乳腺细胞中表达,且具有较好的乳腺特异性,为研发乳腺生物反应器表达载体提供新的方法。     

SARS—CoVSars7a和EGFP融合蛋白真核表达载体构建及其表达

根据SARS-CoV sars7a基因设计并化学合成部分重叠引物,经二轮PCR获得sars7a基因片段,以此片段为模板并利用一对带有Kozak序列及删除终止密码的引物进行PCR,获得产物与pEGFP-N1载体连接,使sars7a基因位于．EGFP的基因上游,得到含编码Sars7a-EGFP融合蛋白基因的哺乳动物细胞表达载体。采用细胞核转染技术将重组表达载体转染K562细胞,以流式细胞仪和共聚焦显微镜分析,可检测到EGFP的绿色荧光,表明Sars7a—EGFP得到表达,该蛋白分布于整个细胞,提示Sars7a并非膜蛋白,更可能是胞浆蛋白。此外,该蛋白的表达对K562细胞凋亡无明显影响。     

Reporter system for the detection of in vivo gene conversion

We have devised a system for the study of in vivo gene correction based on the detection of color variants of the green fluorescent protein (GFP) from the jellyfish Aequorea victoria. The intensity and spectra of the fluorescence emitted by the blue (BFP) and red-shifted (EGFP) variants of GFP differ from each other. We modified one nucleotide from an EGFP expression vector that we predicted would yield a blue variant (TAC-CAC, Tyr⁶⁶-His⁶⁶). Cells that were either transiently or stably transfected with the reporter system were used to test the functionality and feasibility of the detection of in vivo gene correction. A thio-protected single-stranded oligonucleotide designed to convert the genotype of the blue variant to that of the EGFP variant by the correction of a single base pair was delivered to the reported cells using a variety of methodologies and strategies. Conversion events were easily observed using fluorescent microscopy because of the enhanced emission intensity and different spectra of the EGFP variant.