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，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标准化微阵列数据。根据样本之间的差异表达水平、聚类技术和提议的功能，鉴定了几个基因。使用RT-PCR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室对粘附进行定量。 研究发现基因 siat7e 和 lama4 会影响 HeLa 细胞的粘附和形态。在不依赖贴壁的 HeLa 细胞中，降低 siat7e（一种 II 型膜糖基化唾液酸转移酶）的表达会导致更大的聚集和形态变化。当编码分泌糖蛋白的 lama4 的表达增强时，在不依赖贴壁的 HeLa 细胞中也观察到了类似的效果。就生长速率而言，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丝裂原激活蛋白激酶具有序列同源性的蛋白质，被鉴定为 CHO、HEK-293 和 HeLa 细胞中细胞生长的可能增强剂。三个基因已申请临时专利，并且可能会扩大到包括其他基因。 基于上述工作，我们决定将重点放在 MDCK 细胞上。 MDCK 细胞目前被认为是流感病毒繁殖和用于疫苗生产的血凝素 (HA) 生产的含胚卵的替代品。 MDCK 细胞被发现适合病毒生产，但它们无法在悬浮液中生长，这给放大过程带来了负担，从而影响了它们的生产能力。贴壁依赖性 MDCK 细胞被转化为贴壁非依赖性细胞，由于转染人 sia7te 基因 (ST6GalNac V)，能够在悬浮液中生长。当比较贴壁依赖性和非贴壁依赖性 HeLa 细胞的基因转录时，该基因先前被鉴定为在细胞粘附中具有重要作用。与亲本 MDCK 细胞不同，表达 siat7e 的细胞能够以悬浮培养物的形式在摇瓶中生长，达到 7x105 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，表达siat7e的细胞被B型流感/维多利亚/504/2000菌株感染。确定细胞源性病毒保留了与卵源性病毒相似的抗原特性，并且它们的核苷酸序列是相同的。表达siat7e的细胞的血凝素比产量（以每106个细胞的血凝单位表示）比亲本MDCK细胞的比产量高约20倍。如果该悬浮过程扩大规模，则从 10 升 siat7e 表达细胞中以 106 个细胞/ml 的浓度生产 HA 的潜力将相当于从 10,000 个含胚卵中获得的 HA 量。