Dual Cre and Dre recombinases mediate synchronized lineage tracing and cell subset ablation in vivo

Wang, Haixiao; He, Lingjuan; Li, Yan; Pu, Wenjuan; Zhang, Shaohua; Han, Xiao; Lui, Kathy O.; Zhou, Bin

doi:10.1016/j.jbc.2022.101965

Cited by 8 publications

(4 citation statements)

References 57 publications

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“…The first option is to identify genes that are uniquely expressed in cardiac fibroblasts and then generate CreER mouse lines based on them. Another option is to develop a dual recombination system with one recombinase, such as Cre, that expresses specifically in fibroblasts and another recombinase, such as Dre, that expresses specifically in the heart 28,29 . The combination of the two recombinases can achieve heart and fibroblast specificity.…”

Section: Discussionmentioning

confidence: 99%

Heterogeneity and Functional Analysis of Cardiac Fibroblasts in Heart Development

Deng,

He,

et al. 2023

Preprint

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Background As one of the major cell types in the heart, fibroblasts play critical roles in multiple biological processes. Cardiac fibroblasts are known to develop from multiple sources, but their transcriptional profiles have not been systematically compared. Furthermore, while the function of a few genes in cardiac fibroblasts has been studied, the overall function of fibroblasts as a cell type remains uninvestigated. Methods Single-cell mRNA sequencing (scRNA-seq) and bioinformatics approaches were used to analyze the genome-wide genes expression and extracellular matrix genes expression in fibroblasts, as well as the ligand-receptor interactions between fibroblasts and cardiomyocytes. Single molecular in situ hybridization was employed to analyze the expression pattern of fibroblast subpopulation-specific genes. The Diphtheria toxin fragment A (DTA) system was utilized to ablate fibroblasts at each developmental phase. Results Using RNA staining of Col1a1 at different stages, we grouped cardiac fibroblasts into four developmental phases. Through the analysis of scRNA-seq profiles of fibroblasts at 18 stages from two mouse strains, we identified significant heterogeneity, preserving lineage gene expression in their precursor cells. Within the main fibroblast population, we found differential expressions of Wt1, Tbx18, and Aldh1a2 genes in various cell clusters. Lineage tracing studies showed Wt1- and Tbx18-positive fibroblasts originated from respective epicardial cells. Furthermore, using a conditional DTA system-based elimination, we identified the crucial role of fibroblasts in early embryonic and heart growth, but not in neonatal heart growth. Additionally, we identified the zone- and stage-associated expression of extracellular matrix genes and fibroblast-cardiomyocyte ligand-receptor interactions. This comprehensive understanding sheds light on fibroblast function in heart development. Conclusion We observed cardiac fibroblast heterogeneity at embryonic and neonatal stages, with preserved lineage gene expression. Ablation studies revealed their distinct roles during development, likely influenced by varying extracellular matrix genes and ligand-receptor interactions at different stages.

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Section: Discussionmentioning

confidence: 99%

Heterogeneity and Functional Analysis of Cardiac Fibroblasts in Heart Development

Deng,

He,

et al. 2023

Preprint

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“…An analogous approach to lineage-tracing studies, offered by GEMMs, involves the capability to selectively eliminate a specific subset of cells within tumors to interrogate their role in tumor maintenance and progression. Such lineage-ablation experiments commonly utilize a system where a suicide gene, such as the diphtheria toxin receptor (DTR), is placed under the control of gene regulatory elements that exclusively mark a cell type of interest [ 122 ]. Mouse cells exhibit insensitivity to diphtheria toxin (DT), an exceptionally potent cytotoxic peptide, due to the absence of the diphtheria toxin receptor (DTR) expression.…”

Section: Exploring Cancer Heterogeneity: Challenges and Prospects In ...mentioning

confidence: 99%

From Chaos to Opportunity: Decoding Cancer Heterogeneity for Enhanced Treatment Strategies

Ottaiano,

Ianniello,

Santorsola

et al. 2023

Biology

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Cancer manifests as a multifaceted disease, characterized by aberrant cellular proliferation, survival, migration, and invasion. Tumors exhibit variances across diverse dimensions, encompassing genetic, epigenetic, and transcriptional realms. This heterogeneity poses significant challenges in prognosis and treatment, affording tumors advantages through an increased propensity to accumulate mutations linked to immune system evasion and drug resistance. In this review, we offer insights into tumor heterogeneity as a crucial characteristic of cancer, exploring the difficulties associated with measuring and quantifying such heterogeneity from clinical and biological perspectives. By emphasizing the critical nature of understanding tumor heterogeneity, this work contributes to raising awareness about the importance of developing effective cancer therapies that target this distinct and elusive trait of cancer.

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“…Despite its limited application, the Vika/vox system has demonstrated suitability as an alternative or a combinatorial partner with other site-speci c recombinase (SSR) systems in vivo, as demonstrated by Karimova et al 6,7 . The Cre/loxP and Dre/rox systems are widely combined for various purposes as shown by, for instance, in vivo lineage tracing 8,9 . Similarly, the combination of Cre/loxP and Flpo/FRT systems serves as the foundation for gene manipulation using the EUCOMM/KOMP knockout rst alleles 3 .…”

Section: Introductionmentioning

confidence: 99%

Multistep allelic conversion in mouse pre-implantation embryos by AAV vectors

Nickl,

Jenickova,

Elias

et al. 2024

Preprint

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Site-specific recombinases (SSRs) are critical for achieving precise spatiotemporal control of engineered alleles. These enzymes play a key role in facilitating the deletion or inversion of loci flanked by recombination sites, resulting in the activation or repression of endogenous genes, selection markers or reporter elements. However, multiple recombination in complex alleles can be laborious. To improve this, a new and efficient method using AAV vectors can simplify the conversion of systems based on Cre, Flpo, Dre and Vika recombinases. In this study, we present an effective method for ex vivo allele conversion using Cre, Flp (flippase), Dre, and Vika recombinases, employing adeno-associated viruses (AAV) as a delivery vector. AAVs enable efficient allele conversion with minimal toxicity in a reporter mouse line. Moreover, AAVs facilitate sequential allele conversion, essential for fully converting alleles with multiple recombination sites, typically found in conditional knockout mouse models. While simple allele conversions show a 100% efficiency rate, complex multiple conversions consistently achieve an 80% conversion rate. Overall, this strategy markedly reduces the need for animals and significantly speeds up the process of allele conversions, representing a significant improvement in genome engineering techniques.

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Dual Cre and Dre recombinases mediate synchronized lineage tracing and cell subset ablation in vivo

Cited by 8 publications

References 57 publications

Heterogeneity and Functional Analysis of Cardiac Fibroblasts in Heart Development

Heterogeneity and Functional Analysis of Cardiac Fibroblasts in Heart Development

From Chaos to Opportunity: Decoding Cancer Heterogeneity for Enhanced Treatment Strategies

Multistep allelic conversion in mouse pre-implantation embryos by AAV vectors

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