Numerous studies have contributed to the induction of pluripotency in an abundance of cell types; however, transfection techniques and efficiency have yielded undesirable outcomes. Traditionally, the use of viral vectors as a mode of transmission has proven to be efficient in the induction of pluripotency transcription factors in mammalian cells. The increasing concern is random insertion of viral components within the host genome due to the viral mode of replication. The delivery of messenger RNA by cationic lipid delivery vehicles circumvents the viral concerns and provides an efficient and safe mode of reprogramming. Synthetic mRNA can be used to initiate endogenous gene expression while maintaining cellular viability in bovine somatic cells. In this study, bovine fetal fibroblast cells were initially transfected with In Vitro Transcribed (IVT) RNA expressing Green Fluorescent Protein (GFP) to determine adequate transfection parameters. Mammalian expression vectors, encoded with either GFP or pluripotency associated transcription factors OCT4, SOX2, c-MYC, or KLF4, were obtained from a plasmid repository and used as IVT templates. The mRNA was produced in vitro to include a 5 cap as well as a 3 polyA tail in order to mimic in vivo mRNA packaging. Primary cultures of bovine fetal fibroblasts were transfected with ivtRNA by way of a cation lipid delivery vehicle, Lipofectamine, for endocytotic uptake. This process allows the mRNA to bypass the phospholipid bilayer and enter the cell. The incorporation of modified bases during the in vitro transcription process was adopted to reduce cell immune response. Addition of small molecules to enhance the reprogramming process was evaluated as well. The success of ivtRNA transfection in bovine fetal fibroblast cells was determined through the measurement of cellular viability, mean fluorescence by flow cytometry under different concentrations of mRNA, and gene analysis measured by quantitative PCR.
| Identifer | oai:union.ndltd.org:LSU/oai:etd.lsu.edu:etd-11152013-112056 |
| Date | 25 November 2013 |
| Creators | Adams, Tricia |
| Contributors | Cooper, Richard K, Eilertsen, Kenneth J, Lynn, John W, Bondioli, Kenneth R |
| Publisher | LSU |
| Source Sets | Louisiana State University |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.lsu.edu/docs/available/etd-11152013-112056/ |
| Rights | restricted, I hereby certify that, if appropriate, I have obtained and attached herein a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to LSU or its agents the non-exclusive license to archive and make accessible, under the conditions specified below and in appropriate University policies, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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