Background: 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 RNA especially microRNA and siRNA. We initiated this work working with specific microRNAs and we continued performing high throughput techniques, analyzing libraries containing hundreds of microRNAs and thousands of siRNAs. In the first phase, by conducting microRNA analysis, we identified five microRNAs mimics: (hsa-miR-22-5p, hsa-miR-18a-5p, hsa-miR-22-3p, hsa-miR-429 and hsa-miR-2110) that improved functional expression of the tested protein by as much as 48%. In the second phase. by conducing siRNA analysis, of 22000 genes, we identified 10 genes whose inhibition improve recombinant protein expression. Among the 10 confirmed genes, OAZ1-the gene encoding the ornithine decarboxylase antizyme1- was selected for detailed investigation, since its silencing improved the reporter protein production without affecting cell viability. Silencing OAZ1 caused an increase of the ornithine decarboxylase enzyme and the cellular levels of putrescine and spermidine; an indication that increased cellular polyamines enhances luciferase expression without affecting its transcription. The study shows that OAZ1 is a novel target for improved expression of recombinant proteins. The genome-scale screening performed in this work can establish the foundation for targeted design of an efficient mammalian cell platform for various biotechnological applications. Creating HEK cell line in which the OAZ1 gene is not functioning was the next step in this project. By using the crisper technology an HEK cell line lacking OAZ1 was created. The cells were found to have three times increased expression of luciferase, at the same time the cells growth properties and metabolic activities were not affected. Another gene that was among the 10 finalists of the siRNA screening is CASP8AP2. We tested the effect of knocking out this gene on protein expression and the initial results are good. The plan is to create a stable cell line that, like the OAZ1 negative strain, can express higher level of recombinant proteins. Io addition, we demonstrated a novel approach for identifying target genes of microRNA by integrating the differential gene expression analysis with information obtained from our previously-conducted High-Throughput siRNA screening. After identifying the down-regulated genes and cross-referenced them with the predicted targets of the miR-22-3p, they were further cross-referenced with the previously conducted siRNA screen. The identified genes were validated as being involved in improving luciferase expression by using siRNA and qRT-PCR. The HPK1protein was identified as the miRNA target that when repressed, increased luciferase and GPC3 expression by 3.3-fold and 2.2-fold respectively. The work continued by evaluating co-inhibition of target genes, knocking out the target gene by crisper methodology and over expressing the selected microRNA. The lab started to work on clumping issues which occur when cells are propagating in suspension in shake flask and bioreactors. So far, this issue is being dealt by treating the culture with different external means such as adding detergents or modifying mixing strategies and agitation means. However, this plethora of choice reflects a range of sub-optimality instead of solutions that are rooted in specific understanding. Here, we hope to evaluate HEK 293 suspension culture surfaceome features that are responsible for clumping. As a means of screening, we are using a differential clumping model alongside surface biotinylation techniques and proteomics. We hope to validate screen hits with siRNA transfection to bring about transitory knockdown. The long-term goal is to produce an HEK 293 suspension cell line that has good growth characteristics while avoiding clumping in addition to being readily transfected, under a variety of media choices and culture conditions. Less cell clumping should benefit culture reliability and reproducibility as well as transfection reliability and reproducibility. Currently, we are working to refine sample homogeneity, and increase cell viability in the isolation process.
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