Identification of genes realted to spcific properties of mammlian cells

与哺乳动物细胞特定特性相关的基因的鉴定

• 批准号: 7967773
• 负责人: Joseph Shiloach
• 金额: $ 32.36万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7967773
• 关键词: Adhesions; Affect; Algorithms; Base Sequence; Behavior; Biochemical; Bioreactors; Cell Adhesion; Cell Line; Cells; Cyclin-Dependent Kinases; Data; Diagnostic; Enhancers; Gene Expression; Genes; Genetic Transcription; Glycoproteins; Growth; Hela Cells; Hemagglutination; Hemagglutinin; Human; Influenza; Legal patent; MDCK cell; Mammalian Cell; Membrane; Microarray Analysis; Mitochondria; Mitogen-Activated Protein Kinases; Morphology; Phase; Phenotype; Plasmids; Process; Production; Property; Proteins; RNA Interference; Reverse Transcriptase Polymerase Chain Reaction; Role; Sampling; Sequence Homology; Sialyltransferases; Suspension Culture; Suspension substance; Suspensions; Techniques; Therapeutic; Time; Transfection; Vaccines; Virus; Work; base; cell behavior; cell growth; egg; flasks; improved; influenzavirus; research study; scale up

To identify additional genes functionally related to specific cellular properties, cell lines with different phenotypes were grown in bioreactors and sampled for microarray analysis. A combination of data filtering and clustering algorithms was applied to normalize microarray data. Based on the level of differential expression between samples, clustering techniques, and proposed functionality, several genes were identified. The expression of theses genes was verified using RT-PCR. Gene expression levels were altered using RNAi (gene blocking) and plasmids (gene enhancement). Adhesion was quantified using both a cell counter and a shear flow chamber. The genes siat7e and lama4 were found to impact the adhesion and the morphology of HeLa cells. Decreasing the expression of siat7e, a type II membrane glycosylating sialyltransferase, in anchorage-independent HeLa cells resulted in greater aggregation and morphological changes. Similar effects were seen in anchorage-independent HeLa cells when the expression of lama4, which encodes a secreted glycoprotein, was enhanced. In relation to growth rate, two different genes, one encoding a mitochondrial assembly protein and the other encoding a protein with sequence homology to both cyclin-dependent kinases and mitogen-activated protein kinases, were identified as possible enhancers of cellular growth in CHO, HEK-293, and HeLa cells. A provisional patent has been filed for three genes and may be expanded to include other genes. Based on the above work we decided to concentrate on MDCK cells. MDCK cells are currently being considered as an alternative to embryonated eggs for influenza virus propagation and hemagglutinin (HA) production intended for vaccine manufacturing. MDCK cells were found suitable for the virus production but their inability to grow in suspension burdens the process of scale up and hence their production capability. Anchorage-dependent MDCK cells were converted to anchorage-independent cells, capable of growing in suspension as a result of transfection with the human sia7te gene (ST6GalNac V). This gene was previously identified as having an important role in cellular adhesion when the transcriptions of genes from anchorage-dependent and anchorage-independent HeLa cells were compared. Unlike the parental MDCK cells, the siat7e-expressing cells were capable of growing in shake flasks as suspension cultures, achieving maximum concentration of 7x105 cells/ml while keeping close to 100% viability throughout the growth phase. In production experiments, the siat7e-expressing cells were infected with the Influenza B/Victoria/504/2000 strain. It was determined that the cell-derived viruses retained similar antigenic properties as those obtained from egg-derived viruses and their nucleotide sequences were identical. The specific production of hemagglutinin (expressed in hemagglutination units per 106 cells) from the siat7e-expressing cells was approximately 20 times higher than the specific production from the parental MDCK cells. If this suspension process scales up, the production potential of HA from 10 liter of siat7e-expressing cells at a concentration of 106 cells/ml would be equivalent to the amount of HA obtained from 10,000 embryonated eggs.

为了鉴定与特定细胞特性功能相关的其他基因，将具有不同表型的细胞系在生物反应器中生长并取样用于微阵列分析。应用数据过滤和聚类算法的组合来归一化微阵列数据。基于样品之间的差异表达水平、聚类技术和提出的功能，鉴定了几个基因。用RT-PCR方法验证这些基因的表达。使用RNAi（基因阻断）和质粒（基因增强）改变基因表达水平。使用细胞计数器和剪切流动室两者定量粘附。 发现siat 7 e和lama 4基因影响HeLa细胞的粘附和形态。降低siat 7 e，II型膜糖基化唾液酸转移酶，在锚定非依赖性HeLa细胞的表达导致更大的聚集和形态学变化。在锚定非依赖性HeLa细胞中，当编码分泌糖蛋白的lama 4的表达增强时，也观察到类似的效果。关于生长速率，两种不同的基因，一种编码线粒体组装蛋白，另一种编码与细胞周期蛋白依赖性激酶和有丝分裂原活化蛋白激酶具有序列同源性的蛋白，被鉴定为CHO、HEK-293和HeLa细胞中细胞生长的可能增强剂。目前已经为三个基因申请了临时专利，并可能扩大到包括其他基因。 基于上述工作，我们决定集中于MDCK细胞。MDCK细胞目前被认为是鸡胚的替代品，用于流感病毒繁殖和血凝素（HA）生产，预期用于疫苗生产。发现MDCK细胞适合于病毒生产，但它们不能在悬浮液中生长，这给规模扩大的过程以及它们的生产能力带来负担。锚定依赖性MDCK细胞转化为锚定非依赖性细胞，能够在悬浮液中生长，作为转染人sia 7 te基因（ST 6 GalNac V）的结果。该基因以前被确定为具有重要的作用，在细胞粘附时，从锚定依赖性和锚定非依赖性HeLa细胞的基因的转录进行了比较。与亲本MDCK细胞不同，表达siat 7 e的细胞能够在摇瓶中作为悬浮培养物生长，达到7 xl 05个细胞/ml的最大浓度，同时在整个生长期保持接近100%的活力。在生产实验中，用流感病毒B/维多利亚/504/2000株感染siat 7 e表达细胞。经确定，细胞来源的病毒保留了与从蛋来源的病毒获得的病毒相似的抗原特性，并且它们的核苷酸序列相同。siat 7 e表达细胞的血凝素特异性产量（以每106个细胞的血凝单位表示）比亲本MDCK细胞的特异性产量高约20倍。如果该悬浮方法按比例放大，则来自10升浓度为106个细胞/ml的siat 7 e表达细胞的HA生产潜力将相当于从10，000个含胚卵获得的HA量。