liu.seSearch for publications in DiVA
Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • oxford
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Evaluation of 5´- and 3´-UTR Translation Enhancing Sequences to Improve Translation of Proteins in CHO Cells
Linköping University, Department of Physics, Chemistry and Biology, Biotechnology.
2018 (English)Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
Abstract [en]

The purpose of this project was to identify and evaluate nucleotide sequences enhancing translation of proteins in Chinese hamster ovary (CHO) cells. Candidate sequences were placed in the 5´-untranslated region (UTR) or 3´ UTR respectively and evaluated in a CHO-based expression system with a fluorescent Fc-fusion protein as a model protein.Five plasmid vectors were constructed, two of which designed to have a randomized nucleotide library in their 5´ and 3´ UTR respectively, and three of which designed to hold varying repeats of a known enhancing translation (ET) sequence in their 5´ or 3´ UTR. The plasmid constructs were transfected into CHO cells and the protein expression was analyzed both by fluorescence intensity in single cells using flow cytometry and in bulk by monoclonal antibody titer analysis based on Protein A affinity.The main result is that both flow cytometry and titer analysis indicate that insertion of five repeats of the ET in the 5´UTR has a negative effect on protein expression as compared to the control which had no ET repeats. Results related to the insertion of three ETs in the 5´ UTR were ambiguous. The titer analysis indicated that it had a negative effect on the protein expression compared to the control which had no ET repeats, whereas the flow cytometry results suggest that the effect is negligible. Transfection of library plasmids was unsuccessful; hence no library expression analysis results were achieved. Due to the time constraints of the project, the reason for the unsuccessful transfection of library plasmids was not investigated, but the LTX transfection method is stated as a highly plausible cause.Based on the outcome of this study, two recommendations for future work are suggested. The first one is to continue the focus on UTR sequences in terms of library screening, and to improve the method of transfecting library plasmid constructs into CHO cells using lipofection. The second suggestion for further studies is to test different UTR sequence lengths without involving potential ETs, to rule out the effect and positions of the ETs and investigate the expressional effect of UTR length solely.

Place, publisher, year, edition, pages
2018. , p. 44
Keywords [en]
Regulation, Untranslated regions, 5´ UTR, 3´ UTR, mRNA, Expression, Flow cytometry
National Category
Pharmaceutical Biotechnology
Identifiers
URN: urn:nbn:se:liu:diva-150363ISRN: LITH-IFM-A-EX--18/3489--SEOAI: oai:DiVA.org:liu-150363DiVA, id: diva2:1240050
External cooperation
GE Healthcare Biosciences
Subject / course
Biotechnology
Presentation
2018-06-08, Jordan/Fermi, Linköping, 13:00 (Swedish)
Supervisors
Examiners
Available from: 2018-08-21 Created: 2018-08-20 Last updated: 2018-08-21Bibliographically approved

Open Access in DiVA

Master thesis Ellen Einarsson(2630 kB)174 downloads
File information
File name FULLTEXT01.pdfFile size 2630 kBChecksum SHA-512
f72b4018891fe71987983e10e1ccfeef01072e4f84de167dd4262e91e8ee57ffcaf014311e268450af0733e7149186d4255ce7fde90acb43ff61c39e83956818
Type fulltextMimetype application/pdf

Search in DiVA

By author/editor
Einarsson, Ellen
By organisation
Biotechnology
Pharmaceutical Biotechnology

Search outside of DiVA

GoogleGoogle Scholar
Total: 174 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

urn-nbn

Altmetric score

urn-nbn
Total: 635 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • oxford
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf