Gene position affects protein expression in a 2A polycistronic vector system
Public Deposited- Last Modified
- February 26, 2019
- Creator
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Wall, Joseph Blake
- Affiliation: College of Arts and Sciences, Department of Biology
- Abstract
- Cellular reprogramming of fibroblasts to cardiomyocyte-like cells is achieved by the introduction of a combination of cardiac transcription factors. Successful reprogramming requires a stoichiometric balance of these transcriptions factors at the protein level. Traditional methods to introduce exogenous gene expression required multiple plasmids to be transfected into a cell for reprogramming. A problem is that cells hardly take up these reprogramming factor plasmids at optimal stoichiometric ratios, which decreases reprogramming efficiency. Polycistronic vectors are attractive thanks to the ability to introduce one plasmid with desired genes for expression into a cell, as opposed to multiple plasmids. The challenge of polycistronic vectors is that eukaryotic cells do not have the cellular machinery to process polycistronic proteins during translation. However, viral oligopeptides (called 2A peptides) have been identified that enable ribosomes to skip the formation of a certain peptide bond, allowing for “cleavage” of polycistronic peptides. Despite this advantage, widespread adoption of 2A polycistronic vectors has been limited due to the lack of systematic comparison of the different 2A sequences in a polycistronic system. This project sought to characterize the effect of gene position on protein expression in tricistronic 2A constructs containing fluorescent protein (FP) genes in varying positions. To investigate a 2A polycistronic system, mouse embryonic fibroblasts (MEF-T) were infected with retroviruses carrying the polycistronic vector. These transduced cells were then analyzed via fluorescent microscopy and flow cytometry in order to quantify the expression of each fluorescent protein. It was determined that the gene in the first position achieved the highest level of expression, followed by the third position, with the second position yielding the lowest protein expression. Such a finding is important because it not only shows that gene order matters, but it will potentially help further the utilization of 2A polycistronic systems for cellular reprogramming.
- Date of publication
- spring 2017
- Keyword
- DOI
- Resource type
- Rights statement
- In Copyright
- Note
- Funding: None
- Advisor
- Qian, Li
- Degree
- Bachelor of Science
- Academic concentration
- Biology
- Honors level
- Honors
- Degree granting institution
- University of North Carolina at Chapel Hill
- Graduation year
- 2017
- Language
- English
Relations
- Parents:
This work has no parents.
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