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Sci Rep. 2017 May 19;7(1):2193. doi: 10.1038/s41598-017-02460-2.

Systematic comparison of 2A peptides for cloning multi-genes in a polycistronic vector.

Liu Z1,2,3, Chen O1,2,3, Wall JBJ1,2,3, Zheng M1,2,3, Zhou Y1,2,3, Wang L1,2,3, Ruth Vaseghi H1,2,3, Qian L4,5,6, Liu J7,8,9.

Author information

1
Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, NC, 27599, USA.
2
McAllister Heart Institute, University of North Carolina, Chapel Hill, NC, 27599, USA.
3
Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC, 27599, USA.
4
Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, NC, 27599, USA. li_qian@med.unc.edu.
5
McAllister Heart Institute, University of North Carolina, Chapel Hill, NC, 27599, USA. li_qian@med.unc.edu.
6
Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC, 27599, USA. li_qian@med.unc.edu.
7
Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, NC, 27599, USA. jiandong_liu@med.unc.edu.
8
McAllister Heart Institute, University of North Carolina, Chapel Hill, NC, 27599, USA. jiandong_liu@med.unc.edu.
9
Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC, 27599, USA. jiandong_liu@med.unc.edu.

Abstract

Cloning of multiple genes in a single vector has greatly facilitated both basic and translational studies that require co-expression of multiple factors or multi-units of complex protein. Many strategies have been adopted, among which 2A "self-cleaving" peptides have garnered increased interest for their polycistronic nature, small size and high "cleavage" efficiency. However, broad application of 2 A peptides is limited by the lack of systematic comparison of different 2As alone or in combination. Here we characterized the effect of varying gene position and 2As on the expression of proteins encoded in bi-, tri-, or quad-cistronic constructs. Using direct cardiac reprogramming as an example, we further determined the effect of varied 2As on the efficiency of fluorescent cell labeling and cell fate conversion. We found that the expression of fluorophores decreased as it was moved towards the end of the construct while reprogramming was most efficient with the fluorophore at the second position. Moreover, quad-cistronic TPE2A constructs resulted in more efficient reprogramming than 3P2A or PTE2A constructs. We expect that the bi-, tri-, and quad-cistronic vectors constructed here and our results on protein expression ratios from different 2A constructs could serve to guide future utilization of 2A peptides in basic research and clinical applications.

PMID:
28526819
PMCID:
PMC5438344
DOI:
10.1038/s41598-017-02460-2
[Indexed for MEDLINE]
Free PMC Article

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