The production of therapeutic recombinant proteins in heterologous systems has gained significance since the last decade. For recombinant proteins that require post-translational modifications (PTMs), mammalian systems are preferred. Chinese hamster ovary (CHO) cells are the mammalian cells of choice for production of recombinant proteins. This is because of their ability to provide correct protein-folding and post-translational modifications, displaying high productivity at large scale, ability to grow in suspension mode at high densities in a serum-free media, incapable of infection by most viruses and their history of regulatory approvals. There is an established state of the art technology for development of CHO cells for recombinant protein production. This technology relies on random integration of the gene of interest and gene amplification process for obtaining high expressing clones. There is a high degree of clonal heterogeneity and instability observed in the screened clones. To overcome the process of random integration, this report describes a lentivirus based screening for search of stable and high expressing integration sites in CHO cells. The integration sites are identified by using nrLAM-PCR (non-restrictive linear amplification mediated PCR) coupled with high throughput sequencing. Lentivirus are chosen as they preferentially integrate within the coding regions rendering the possibility of obtaining stable and high expressing clones. In addition, lentivirus vector is designed to possess landing pad for recombinase mediated cassette exchange of viral sequence with foreign DNA. The report describes a successful cassette exchange reaction but with low efficiency. Genome engineering technologies such as CRISPR/Cas, TALENs can used for targeted gene insertion at integration sites and thus establishing stable and efficient production of recombinant proteins in CHO cells. Additionally, an approach for designing synthetic promoters based on Ef1α promoter architecture has been shown. Synthetic promoters are useful for expression of multi-gene cassettes as they are short in length and provide comparable expression levels to the native mammalian promoter.
| Identifer | oai:union.ndltd.org:UPSALLA1/oai:DiVA.org:uu-309017 |
| Date | January 2016 |
| Creators | Doshi, Jiten |
| Publisher | Uppsala universitet, Institutionen för biologisk grundutbildning, ETH Zurich |
| Source Sets | DiVA Archive at Upsalla University |
| Language | English |
| Detected Language | English |
| Type | Student thesis, info:eu-repo/semantics/bachelorThesis, text |
| Format | application/pdf |
| Rights | info:eu-repo/semantics/openAccess |
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