Identification of genes related to spcific properties of mammalian cells

与哺乳动物细胞特定特性相关的基因的鉴定

• 批准号: 10250250
• 负责人: Joseph Shiloach
• 金额: $ 56.1万
• 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/10250250
• 关键词: Affect; Automation; Behavior; Biochemical; Biological Assay; Biotechnology; CDKN2A gene; CRISPR/Cas technology; Cell Cycle Regulation; Cell Line; Cell Survival; Cells; Diagnostic; Engineering; Foundations; Gene Expression; Genes; Growth; Human; Investigation; Knock-out; Libraries; Liquid substance; Luciferases; Mammalian Cell; Metabolic; MicroRNAs; Ornithine Decarboxylase; Plasmids; Production; Property; Proteins; Putrescine; Reagent; Recombinant Proteins; Recombinant adeno-associated virus (rAAV); Recombinants; Reporter; Research; Signal Transduction; Small Interfering RNA; Source; Spermidine; Structure; Techniques; Therapeutic; Time; Transfection; Transgenes; Untranslated RNA; Up-Regulation; Viral; Virus; Work; base; cell behavior; cell growth; density; design; experience; genome editing; genome-wide; improved; novel; protein expression; screening; transcriptome

Background: Mammalian cells are currently the main producers of various biochemical compounds needed for human therapeutic and for diagnostic purposes. These cells have distinct properties, such as slow growth rate and anchorage-dependent behavior, which affect production efficiency. Our research work is concentrating on identification of genes and microRNA that affects production properties. By utilizing these genes and microRNAs it be likely possible to change the cells behavior and improve their production capability. In the last several years we demonstrated that it is possible to improve mammalian cells growth and recombinant protein production by manipulating gene expression using noncoding RNAs, especially microRNAs and siRNAs. We initiated this approach working with specific microRNAs and we continued performing high throughput techniques, analyzing libraries containing hundreds of microRNAs and thousands of siRNAs. By conducing high throughput siRNA analysis of 22000 genes, 10 genes whose inhibition improved recombinant protein expression were identified. Among them OAZ1-the gene encoding the ornithine decarboxylase antizyme1- which was selected for detailed investigation, since its silencing improved the reporter protein production without affecting cell viability. Silencing OAZ1 caused an increase of ornithine decarboxylase expression with increased cellular levels of putrescine and spermidine. The study demonstrated that OAZ1 is a novel target for improved expression of recombinant proteins. The genome-scale screening performed in this work can establish a foundation for targeted design of an efficient mammalian cell platform for various biotechnological applications. Creating HEK cell line in which the OAZ1 gene was deleted was the next step in this project. By using Crispr technology HEK cell line lacking OAZ1 was created, that showed three times increased expression of luciferase without affecting growth and metabolic activities. The work continued by deleting the CASP8AP2 gene that was included in the 10 genes list. We validate the CASP8AP2 gene as an engineering target in HEK293 cells by knocking it out using CRISPR/Cas9 genome editing and assessing the effect of its knockout on recombinant protein expression, cell growth, cell viability, and overall gene expression. HEK293 cells lacking CASP8AP2 showed 7-fold increase in specific expression of recombinant luciferase and a 2.5-fold increase in specific expression of recombinant SEAP, without significantly affecting cell growth and viability. Transcriptome analysis revealed that de-regulation of the cell cycle, specifically the upregulation of the cyclin dependent kinase inhibitor 2A (CDKN2A) gene, contributed to the improvement in recombinant protein expression in CASP8AP2 deficient cells. The results validate the CASP8AP2 gene is a viable engineering target for improved recombinant protein expression in the HEK293 cell line. In addition to protein expression Mammalian cells are the source for virus expression. So, based on our experience utilizing noncoding RNA for improved protein production we looked to identify miRNA or siRNA species to co-express alongside viral structural and helper proteins to affect an increase in rAAV titer, independent of the transgene being delivered. We expect viral production to benefit from a different optimized miRNA profile. Our work thus far has been the refining of screening conditions. The screen can be considered as separate transfection/production and a transduction/assay step. Transfection parameters (plating density, plasmid concentration, transfection reagent, and days between reverse and forward transfection) were chosen to optimize (i) fluorescent protein expression from the ITRs in 293T/17 cells as well as (ii) corresponding transduction seen in COS7 cells also assessed via fluorescent protein expression. Complementarily, transduction parameters were chosen to allow for (i) the highest fluorescent signal given the addition of the same supernatant batches, (ii) minimizing liquid handling error, and (iii) for a level of MOI/signal significantly less than non- saturating. Examples of relevant parameters include HEK293T/17 cell plating density (3e3), total plasmid amount (0.12 g), the reverse and forward transfection reagents (Dharmafect4 and Viafect), and the cell line to evaluate transduction (COS7). Based on initial results, efforts to improve the signal and minimize variability have included increasing the time between reverse and forward transfection, adding more automation steps, moving to luciferase as the assay marker, and adding new specific siRNA libraries to the screen.

背景：目前，哺乳动物细胞是人类治疗和用于诊断目的所需的各种生化化合物的主要生产者。这些细胞具有不同的特性，例如生长速度缓慢和锚定依赖性行为，这会影响生产效率。我们的研究工作集中在影响生产特性的基因和microRNA的鉴定上。通过利用这些基因和microRNA，有可能改变细胞行为并提高其生产能力。 在过去的几年中，我们证明，通过使用非编码RNA，尤其是microRNA和siRNA来操纵基因表达，可以通过操纵基因表达来改善哺乳动物细胞的生长和重组蛋白。我们启动了与特定microRNA一起使用的方法，并继续执行高吞吐量技术，分析包含数百个microRNA和数千个siRNA的库。通过对22000个基因的高吞吐量siRNA分析，鉴定出10个抑制作用改善重组蛋白表达的基因。其中包括OAZ1 - 编码鸟氨酸脱羧酶抗酶1-的基因，该基因被选为详细研究，因为它的沉默改善了报告基因的蛋白质产生而不会影响细胞活力。沉默的OAZ1导致鸟氨酸脱羧酶表达的增加，并随着细胞的腐蚀剂和精子素水平升高。研究表明，OAZ1是改善重组蛋白表达的新靶标。在这项工作中进行的基因组规模筛查可以为各种生物技术应用的有效哺乳动物细胞平台的靶向设计建立基础。创建HEK细胞系列是该项目的下一步。通过使用CRISPR技术，创建了缺乏OAZ1的HEK细胞系，这表明荧光素酶表达增加了三倍，而不会影响生长和代谢活动。通过删除包含在10个基因列表中的CASP8AP2基因来继续工作。我们通过使用CRISPR/CAS9基因组编辑将CASP8AP2基因验证为HEK293细胞中的工程靶标，并评估其敲除对重组蛋白表达，细胞生长，细胞生存力和整体基因表达的影响。缺乏CASP8AP2的HEK293细胞在重组荧光素酶的特异性表达中增加了7倍，重组SEAP的特异性表达增加了2.5倍，而没有显着影响细胞生长和活力。转录组分析表明，细胞周期的消除，特别是细胞周期依赖性激酶抑制剂2a（CDKN2A）基因的上调，有助于改善CASP8AP2缺乏细胞中重组蛋白表达的改善。结果验证CASP8AP2基因是改善HEK293细胞系中重组蛋白表达的可行工程目标。 除蛋白质表达外，哺乳动物细胞是病毒表达的来源。因此，基于我们利用非编码RNA改善蛋白质产生的经验，我们希望鉴定miRNA或siRNA物种与病毒结构和辅助蛋白一起共表达，以影响Raav滴度的增加，而与所传递的转基因无关。我们预计病毒产生将从不同的优化miRNA概况中受益。迄今为止，我们的工作是筛查条件的完善。屏幕可以视为单独的转染/生产和转导/测定步骤。选择转染参数（电平密度，质粒浓度，转染试剂以及反向转染和正向转染之间的天数）以优化（i）（i）293T/17细胞中ITR的荧光蛋白表达以及（ii）在COS7细胞中在COS7细胞中看到的相应转导的（II）也通过荧光蛋白表达进行了评估。补充，选择转导参数以允许（i）添加相同的上清液批次，（ii）最小化液体处理误差的最高荧光信号，以及（iii）MOI/信号的水平明显小于非饱和度。 相关参数的示例包括HEK293T/17细胞电镀密度（3E3），总质粒量（0.12 g），反向和正向转染试剂（Dharmafect4和Viafect）以及用于评估转移（COS7）的细胞系。基于初始结果，改善信号和最小化变异性的努力包括增加反向转染和正向转染之间的时间，添加更多的自动化步骤，将其转移到荧光素酶作为测定标记，并将新的特定siRNA库添加到屏幕上。