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A Split-Cre system designed to detect simultaneous expression of two genes based on SpyTag/SpyCatcher conjugation and Split-GFP dimerization.
- Source :
-
The Journal of biological chemistry [J Biol Chem] 2021 Oct; Vol. 297 (4), pp. 101119. Date of Electronic Publication: 2021 Aug 25. - Publication Year :
- 2021
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Abstract
- The Split-Cre system is a powerful tool for genetic manipulation and can be used to spatiotemporally control gene expression in vivo. However, the low activity of the reconstituted NCre/CCre recombinase in the Split-Cre system limits its application as an indicator of the simultaneous expression of a pair of genes of interest. Here, we describe two approaches for improving the activity of the Split-Cre system after Cre reconstitution based on self-associating split GFP (Split-GFP) and SpyTag/SpyCatcher conjugation. First, we created the Split-GFP-Cre system by constructing fusion proteins of NCre and CCre with the N-terminal and C-terminal subunits of GFP, respectively. Reconstitution of Cre by GFP-mediated dimerization of the two fusion proteins resulted in recombinase activity approaching that of full-length Cre in living cells. Second, to further increase recombinase activity at low levels of Split-Cre expression, the Split-Spy-GCre system was established by incorporating the sequences for SpyTag and SpyCatcher into the components of the Split-GFP-Cre system. As anticipated, covalent conjugation of the SpyTag and SpyCatcher segments improved Split-GFP dimerization to further increase Cre recombinase activity in living cells. The increased efficiency and robustness of this dual-split system (Split-Cre and Split-GFP) minimize the problems of incomplete double gene-specific KO or low labeling efficiency due to poor NCre/CCre recombinase activity. Thus, this Split-Spy-GCre system allows more precise gene manipulation of cell subpopulations, which will provide advanced analysis of genes and cell functions in complex tissue such as the immune system.<br />Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article.<br /> (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)
- Subjects :
- Protein Multimerization
Escherichia coli genetics
Escherichia coli metabolism
Gene Expression
Green Fluorescent Proteins biosynthesis
Green Fluorescent Proteins genetics
Integrases genetics
Integrases metabolism
Microorganisms, Genetically-Modified genetics
Microorganisms, Genetically-Modified metabolism
Recombinant Fusion Proteins biosynthesis
Recombinant Fusion Proteins genetics
Subjects
Details
- Language :
- English
- ISSN :
- 1083-351X
- Volume :
- 297
- Issue :
- 4
- Database :
- MEDLINE
- Journal :
- The Journal of biological chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 34450162
- Full Text :
- https://doi.org/10.1016/j.jbc.2021.101119