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Review
. 2023 May;56(5):e13446.
doi: 10.1111/cpr.13446. Epub 2023 Apr 14.

Perfect duet: Dual recombinases improve genetic resolution

Hongxin Li  1 , Wendong Weng  1 , Bin Zhou  1   2   3   4
Affiliations
Review

Perfect duet: Dual recombinases improve genetic resolution

Hongxin Li et al. Cell Prolif. 2023 May.

Abstract

As a powerful genetic tool, site-specific recombinases (SSRs) have been widely used in genomic manipulation to elucidate cell fate plasticity in vivo, advancing research in stem cell and regeneration medicine. However, the low resolution of conventional single-recombinase-mediated lineage tracing strategies, which rely heavily on the specificity of one marker gene, has led to controversial conclusions in many scientific questions. Therefore, different SSRs systems are combined to improve the accuracy of lineage tracing. Here we review the recent advances in dual-recombinase-mediated genetic approaches, including the development of novel genetic recombination technologies and their applications in cell differentiation, proliferation, and genetic manipulation. In comparison with the single-recombinase system, we also discuss the advantages of dual-genetic strategies in solving scientific issues as well as their technical limitations.

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Conflict of interest statement

No conflict of interest.

Figures

FIGURE 1
FIGURE 1
Mechanisms and examples of OR‐logic actualization. (A,B) Kit‐CreER is active in both non‐CMs and CMs. To block Kit‐CreER labelling in CMs, constitutive Tnni3‐Dre induces Dre‐rox recombination and removes the loxP site for Cre‐recombination. (C,D) The working principle of the nested reporter (NR1) is that the inducible Dre‐mediated recombination removes the Cre‐induced ZsGreen expression, which allows the precise labelling of BECs without targeting any hepatocytes. (E,F) IR1 is used to precisely label pancreatic ductal cells without contaminating acinar cells.
FIGURE 2
FIGURE 2
Mechanisms and examples of AND‐logic actualization. (A) The mechanistic illustration shows that only in BASCs where Sftpc‐DreER and Scgb1a1‐CreER are both active, tdT expression is induced. (B) Bronchioalveolar stem cells contribute to bronchial and alveolar cells in different scenarios. (C) A tandem reporter with ZsGreen and tdTomato induced by Dre and Cre‐mediated recombination, respectively. (D,E) Strategies for tracing PDGFRa+ Sca1+ cells and PDGFRb+ Sca1+ cells. (F) PDGFRa+ Sca1+ cells contribute to vascular SMCs in injury.
FIGURE 3
FIGURE 3
Mechanisms and examples of inducible capturing of transient gene activation. Using (A) Alb‐DreER or (B) AAV9‐Dre/Tnnt2‐DreER, Ki67 activation can be recorded in hepatocytes or cardiomyocytes upon tamoxifen/AAV administration. (C) Proliferation spatial pattern is shown in the heart. (D) Inducible and continuous recording of gene activation without multiple drug administration. (E) Using EMT‐gene‐LSL‐Dre to explore the role of genes in EMT. (F) N‐Cad‐activated, but not Vimentin‐activated breast cancer cells predominantly colonize the lung. (G,H) Cre‐ and Flp‐induced dual‐recombinase system detecting EMT in pancreatic carcinoma.
FIGURE 4
FIGURE 4
Mechanisms and examples of genetic manipulation with dual recombinases. (A) To activate Cre in WAT but not BAT, Plin1‐dCreER is designed to allow UCP1‐Dre to remove the CreER sequence in BAT. (B) Prox1‐RSR‐CreER is designed to be activated by endothelial‐specific Cdh5‐Dre. (C) With IR1‐DTR, beta cells can be ablated after DT administration, and ZsGreen+ non‐beta cells can transdifferentiate into insulin‐expressing cells.
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