Medical College of Wisconsin
CTSICores SearchResearch InformaticsREDCap

Systematic comparison of 2A peptides for cloning multi-genes in a polycistronic vector. Sci Rep 2017 May 19;7(1):2193

Date

05/21/2017

Pubmed ID

28526819

Pubmed Central ID

PMC5438344

DOI

10.1038/s41598-017-02460-2

Scopus ID

2-s2.0-85019896240   242 Citations

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‚ÄČA 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.

Author List

Liu Z, Chen O, Wall JBJ, Zheng M, Zhou Y, Wang L, Vaseghi HR, Qian L, Liu J

Author

Ziqing Liu PhD Assistant Professor in the Physiology department at Medical College of Wisconsin




MESH terms used to index this publication - Major topics in bold

Animals
Cell Line
Cell Tracking
Cellular Reprogramming Techniques
Gene Expression
Gene Order
Genetic Vectors
Humans
Mice
Peptides
Promoter Regions, Genetic
Tandem Repeat Sequences