AbstractTo understand gene function, it is necessary to compare cells carrying the mutated target gene with normal cells. In most biomedical studies, the cells being compared are in different mutant and control animals and, therefore, do not experience the same epigenetic changes and tissue microenvironment.

摘要为了了解基因功能，有必要将携带突变靶基因的细胞与正常细胞进行比较。。

The experimental induction of genetic mosaics is essential to determine a gene cell-autonomous function and to model the etiology of diseases caused by somatic mutations. Current technologies used to induce genetic mosaics in mice lack either accuracy, throughput or barcoding diversity. Here we present the iFlpMosaics toolkit comprising a large set of new genetic tools and mouse lines that enable recombinase-dependent ratiometric induction and single-cell clonal tracking of multiple fluorescently labeled wild-type and Cre-mutant cells within the same time window and tissue microenvironment.

遗传镶嵌的实验诱导对于确定基因细胞自主功能和模拟由体细胞突变引起的疾病的病因至关重要。目前用于诱导小鼠遗传镶嵌的技术缺乏准确性，吞吐量或条形码多样性。在这里，我们介绍了iFlpMosaics工具包，该工具包包含大量新的遗传工具和小鼠品系，可在同一时间窗口和组织微环境中对多个荧光标记的野生型和Cre突变细胞进行重组酶依赖性比率诱导和单细胞克隆追踪。

The labeled cells can be profiled by multispectral imaging or by fluorescence-activated flow cytometry and single-cell RNA sequencing. iFlpMosaics facilitate the induction and analysis of genetic mosaics in any quiescent or progenitor cell, and for any given single or combination of floxed genes, thus enabling a more accurate understanding of how induced genetic mutations affect the biology of single cells during tissue development, homeostasis and disease..

标记的细胞可以通过多光谱成像或荧光激活流式细胞术和单细胞RNA测序进行分析。iFlpMosaics有助于诱导和分析任何静止或祖细胞中的遗传镶嵌，以及任何给定的单个或组合的floxed基因，从而能够更准确地了解诱导的基因突变如何在组织发育，体内平衡和疾病过程中影响单细胞的生物学。。

MainThe induction of a gene deletion or mutation can substantially alter a cell’s phenotype, and over time, it can also affect the surrounding tissue’s phenotype. Scientists often analyze mutant cells or tissues that have carried genetic mutations for several days, months or even years and compare their phenotype with that of independent control cells or tissues from distinct nonmutant animals.

主要基因缺失或突变的诱导可以显着改变细胞的表型，随着时间的推移，它也可以影响周围组织的表型。科学家经常分析携带基因突变数天、数月甚至数年的突变细胞或组织，并将其表型与来自不同非突变动物的独立对照细胞或组织的表型进行比较。

During the process from gene mutation to phenotypic manifestation and analysis, the biology of the targeted and surrounding tissue often undergoes significant changes. Since the wild-type cells surrounding mutant cells are themselves a source of biochemical factors, any alteration to their development or function by the mutant cells will trigger changes in a key tissue feedback mechanism, and any such changes will impact the phenotype of the mutant cells in a noncell-autonomous manner, that is, not directly dependent of the initially induced genetic mutation itself1,2,3.

在从基因突变到表型表现和分析的过程中，目标组织和周围组织的生物学通常会发生重大变化。由于突变细胞周围的野生型细胞本身就是生化因子的来源，突变细胞对其发育或功能的任何改变都会触发关键组织反馈机制的变化，任何此类变化都会影响突变细胞的表型。以非细胞自主的方式，即不直接依赖于最初诱导的基因突变本身1,2,3。

Over time, this phenomenon often generates secondary mutant tissue phenotypes that can confound interpretation of the primary impact of a gene mutation on a cell’s phenotype.Genetic mosaics are a powerful research tool because they allow the study of cell-autonomous gene function when mutant and wild-type cells originate from the same progenitor cells.

随着时间的推移，这种现象通常会产生次级突变组织表型，这可能会混淆基因突变对细胞表型的主要影响的解释。遗传镶嵌是一种强大的研究工具，因为当突变型和野生型细胞来自同一祖细胞时，它们可以研究细胞自主基因的功能。

In this scenario, the only difference between the cells being compared is the induced mutation, in an otherwise identical organism, genetic background and tissue microenvironment. Mouse models that allow the timed induction of somatic genetic mosaics are essential to accurately understand a gene function and model biological or disease processes caused by sporadic somatic mutations.In Drosophila, interchromosomal mitotic recombination associated with distinct tissu.

在这种情况下，被比较的细胞之间的唯一区别是在其他相同的生物体，遗传背景和组织微环境中诱导的突变。允许定时诱导体细胞遗传镶嵌的小鼠模型对于准确理解基因功能和模拟由散发性体细胞突变引起的生物学或疾病过程至关重要。在果蝇中，染色体间有丝分裂重组与不同的组织有关。

iDre/Flp

iDre/Flp

Progenitor enables the induction of twin-spot clonesThe results above show that there is substantial intercellular clonal variability when different progenitor cells, occupying different tissue locations, are induced. The iFlpMosaics technology shown above can only be induced in distinct progenitor cells, and therefore, it requires averaging of the clonal expansion data from many different independently labeled progenitor cells.

。上面显示的iFlpMosaics技术只能在不同的祖细胞中诱导，因此，它需要平均来自许多不同独立标记的祖细胞的克隆扩增数据。

By contrast, The MADM approach allows the generation of labeled wild-type and mutant cells from the same progenitor cell (twin-spot clones) and, in this way, gives a very precise estimate of how a gene mutation impacts the mobilization and proliferation phenotypes of a single-cell derived progeny. However, as mentioned above, MADM is a cumbersome method and cannot be effectively induced at a specific timepoint with CreERT2.To overcome the limitations of current approaches to understanding the role of genes in single-progenitor cell biology, we designed the iDre/FlpProgenitor allele (Fig.

。然而，如上所述，MADM是一种麻烦的方法，不能在CreERT2的特定时间点有效诱导。为了克服目前理解基因在单祖细胞生物学中作用的方法的局限性，我们设计了iDre/FlpProgenitor等位基因（图。

5a). This allele can be induced by both DreERT2 or FlpO-ERT2. Therefore, we also generated a new Tg(Ins-CAG-DreERT2) allele by using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) assisted targeting into the preexisting and screened Tg(Ins-CAG-FlpO-ERT2) allele (Fig.

5a）。该等位基因可以由DreERT2或FlpO-ERT2诱导。因此，我们还通过使用聚集的规则间隔短回文重复序列（CRISPR）和CRISPR相关蛋白9（Cas9）辅助靶向预先存在和筛选的Tg（Ins-CAG-FlpO-ERT2）等位基因产生了一个新的Tg（Ins-CAG-DRERT2）等位基因（图）。

5b), which allowed us to induce much higher frequencies of recombination (Fig. 5c). The iDre/FlpProgenitor allele also enabled us to increase the sensitivity and recombination efficiency of all FlpO-ERT2 lines. With the Cdh5-FlpO-ERT2 line, we increased EC-recombination efficiency 14-fold, recombining 51% of all ECs (Fig.

5b），这使我们能够诱导更高频率的重组（图5c）。iDre/FlpProgenitor等位基因还使我们能够提高所有FlpO-ERT2系的敏感性和重组效率。使用Cdh5-FlpO-ERT2系，我们将EC重组效率提高了14倍，重组了51%的EC（图）。

5d). Recombination efficiency after combining iDre/FlpProgenitor with the Tg(Ins-CAG-FlpO-ERT2) allele was increased 25-f.

5d）。将iDre/FlpProgenitor与Tg（Ins-CAG-FlpO-ERT2）等位基因结合后，重组效率提高了25-f。

Data availability

数据可用性

The RNA-seq data can be viewed at the Gene Expression Omnibus under accession number GSE257723. The instructions and code to reproduce all scRNA-seq or image analysis results can be found at GitHub via https://github.com/RuiBenedito/Benedito_Lab/tree/main/iFlpMosaics. The unprocessed FACS raw data files or original microscopy images of the data are available upon request.

RNA-seq数据可以在Gene Expression Omnibus上查看，登录号为GSE257723。复制所有scRNA-seq或图像分析结果的说明和代码可以在GitHub上通过https://github.com/RuiBenedito/Benedito_Lab/tree/main/iFlpMosaics.未经处理的FACS原始数据文件或数据的原始显微镜图像可应要求提供。

All other data supporting the findings in this study are included in the main article and associated files. Source data are provided with this paper..

支持本研究结果的所有其他数据均包含在主要文章和相关文件中。本文提供了源数据。。

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Download referencesAcknowledgementsThe research in Rui Benedito laboratory was supported by the European Research Council Starting Grant AngioGenesHD (638028), the European Research Council Consolidator Grant AngioUnrestUHD (101001814), the Ministerio de Ciencia, Innovación y Universidades (SAF2017-89299-P and PID2020-120252RB-I00) and ‘la Caixa’ Banking Foundation (project code HR19-00120 and HR22-00316 AngioHeart) awarded to R.B.

下载参考文献致谢Rui Benedito实验室的研究得到了欧洲研究理事会启动基金AngioGenesHD（638028），欧洲研究理事会合并基金Angiounstuhd（101001814），创新大学科学部（SAF2017-89299-P和PID2020-120252RB-I00）和“la Caixa”银行基金会（项目代码HR19-00120和HR22-00316 AngioHeart）的支持。

The CNIC is supported by the Instituto de Salud Carlos III, the Ministerio de Ciencia, Innovación y Universidades (MICIU) and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (grant CEX2020-001041-S funded by MICIU/AEI/10.13039/501100011033). The microscopy experiments were performed in the CNIC Microscopy and Dynamic Image Unit, an ICTS-ReDib co-funded by MCIN (/AEI/10.13039/501100011033) and the EDRF ‘A Way to Build Europe’ (number ICTS-2018-04-CNIC-16).

网络中心得到了萨卢德·卡洛斯三世研究所、科学部、创新大学（MICIU）和亲网络中心基金会的支持，是塞韦罗·奥乔亚卓越中心（grant CEX2020-001041-S，由MICIU/AEI/10.13039/501100011033资助）。显微镜实验在CNIC显微镜和动态图像部门进行，该部门是由MCIN（/AEI/10.13039/501100011033）和EDRF“建设欧洲的方式”（编号ICTS-2018-04-CNIC-16）共同资助的ICTS ReDib。

I.G.-G. was supported by a PhD fellowship from Fundación La Caixa (CX-SO-16-1). A.R. was supported by The Youth Employment Initiative PEJD-2019-PRE/BMD-16990. L.G.-O. was supported by the Spanish Ministry of Economy and Competitiveness (PRE2018-085283). S.G. was supported by a Juan de la Cierva Fellowship (FJC2020-044237-I).

一、 。A、 R.得到了青年就业倡议PEJD-2019-PRE/BMD-16990的支持。五十、 G.-O.得到了西班牙经济和竞争力部的支持（PRE2018-085283）。S、 G.得到了Juan de la Cierva奖学金（FJC2020-044237-I）的支持。

M.F.-C. was supported by PhD fellowships from Fundación La Caixa (CX_E-2015-01). We thank S. Bartlett (CNIC) for English editing and the members of the CNIC transgenesis, microscopy, genomics, cytometry and bioinformatics units. We also thank F. Alt (Boston Children’s Hospital, Harvard Medical School), T.

M、 F.-C.得到了Caixa基金会（CX\U E-2015-01）的博士研究金的支持。我们感谢S.Bartlett（CNIC）的英文编辑以及CNIC转基因，显微镜，基因组学，细胞计数和生物信息学部门的成员。我们还要感谢F.Alt（哈佛医学院波士顿儿童医院），T。

Honjo (Kyoto University Institute for Advanced Studies), F. Radtke (Swiss Institute for Experimental Cancer Research), R. H. Adams (Max Planck Institute for Molecular Biomedicine) and R. De Pinho (MD Anderson Cancer Center) for sharing.

Honjo（京都大学高级研究所），F.Radtke（瑞士实验癌症研究所），R.H.Adams（马克斯·普朗克分子生物医学研究所）和R.De Pinho（MD安德森癌症中心）分享。

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PubMed Google ScholarContributionsI.G.-G. and R.B. designed most of the experiments, interpreted results, assembled the figures and wrote the paper. R.B. designed all DNA vectors used for transgenesis. I.G.-G. did most of the DNA engineering (cloning), CRISPR–Cas9 genome targeting, animal experiments, confocal microscopy, FACS and scRNA-seq analysis.

PubMed谷歌学术贡献。G、 -G.和R.B.设计了大多数实验，解释了结果，组装了数字并撰写了论文。R、 B.设计了用于转基因的所有DNA载体。一、 G.-G.进行了大部分DNA工程（克隆），CRISPR-Cas9基因组靶向，动物实验，共聚焦显微镜，FACS和scRNA-seq分析。

The mice were generated by the CNIC transgenesis unit. S.G. developed new methods for iFlpMosaics induction, tissue immunostaining and multispectral microscopy imaging, performed animal experiments and image analysis and interpreted results. S.F.R. performed immunostainings, microscopy, qRT-PCR and FACS analysis, image quantifications in Fiji and GraphPad, edited text and figures and assembled figures.

小鼠由CNIC转基因单位产生。S、 G.开发了iFlpMosaics诱导，组织免疫染色和多光谱显微镜成像的新方法，进行了动物实验和图像分析，并解释了结果。S、 F.R.进行了免疫染色，显微镜检查，qRT-PCR和FACS分析，在Fiji和GraphPad中进行了图像定量，编辑了文本和数字以及组装的数字。

L.G.-O, W.L., I.Z., M.D.-L. and M.F.-C. performed animal experiments, FACS, histology and confocal imaging. A.R. cloned and validated the iDre/FlpProgenitor allele. I.G.-G., A.R., C.T. and F.S.-C. analyzed the scRNA-seq data. M.L., M.S.S.-M., A.G.-C., F.L. and V.C.-G. gave general technical assistance with experiments and genotyped the mouse colonies.

五十、 G.-O，W.L.，I.Z.，M.D.-L.和M.F.-C.进行了动物实验，FACS，组织学和共聚焦成像。A、 R.克隆并验证了iDre/FlpProgenitor等位基因。一、 G.G.，A.R.，C.T.和F.S.C.分析了scRNA-seq数据。M、 L.，M.S.S.-M.，A.G.-C.，F.L.和V.C.-G.在实验中提供了一般技术援助，并对小鼠菌落进行了基因分型。

All authors approved the final version of the paper.Corresponding authorCorrespondence to.

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Nature Methods thanks the anonymous reviewers for their contribution to the peer review of this work. Primary Handling Editor: Madhura Mukhopadhyay, in collaboration with the Nature Methods team.

Nature Methods感谢匿名审稿人对这项工作的同行评审做出的贡献。主要处理编辑：Madhura Mukhopadhyay，与Nature Methods团队合作。

Additional informationPublisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Extended dataExtended Data Fig. 1 False positives and false negatives with Cre-dependent mosaic genetics.a, Schematic showing how a standard Cre-reporter can recombine and label cells without the deletion of any floxed gene (G) (false positives) and how the floxed gene can be deleted in non-Cre-recombined cells (false negatives).

Additional informationPublisher的注释Springer Nature在已发布的地图和机构隶属关系中的管辖权主张方面保持中立。扩展数据扩展数据图1 Cre依赖性镶嵌遗传学的假阳性和假阴性。a，示意图显示了标准Cre报告基因如何重组和标记细胞而不删除任何floxed基因（G）（假阳性）以及如何在非Cre重组细胞中删除floxed基因（假阴性）。

b, Different recombination efficiencies of different Rosa26 Cre-reporters in postnatal day 7 mice having the Cdh5-CreERT2 allele, despite all localizing to the Rosa26 locus and having a similar genetic distance between LoxP sites. c,d, Analysis of FACS or immunohistochemistry data reveals that Cre-reporters only accurately report recombination of themselves, not that of other floxed alleles (reporters), particularly at low tamoxifen doses.

b、 不同Rosa26-Cre报告基因在出生后第7天具有Cdh5-CreERT2等位基因的小鼠中的重组效率不同，尽管它们都位于Rosa26基因座上，并且LoxP位点之间的遗传距离相似。c、 d，FACS或免疫组织化学数据的分析表明，Cre报告基因仅准确报告自身的重组，而不是其他floxed等位基因（报告基因）的重组，特别是在低他莫昔芬剂量下。

Data are presented as mean values +/− SD.Source dataExtended Data Fig. 2 iFlpMosaics are neither toxic nor leaky.a, b, Schematic diagrams of the novel Rosa26-iFlpMTomato-Cre/MYFP and Tg-iFlpMTomato-H2B-GFP-Cre/MYFP-H2B-Cherry-FlpO alleles, showing the genetic distances between the mutually exclusive FRT site pairs and the expected outcomes after FlpO/FlpO-ERT2 recombination.

数据以平均值+/-SD表示。源数据扩展数据图2 iFlpMosaics既没有毒性也没有泄漏。a，b，新型Rosa26 iFlpMTomato-Cre/MYFP和Tg-iFlpMTomato-H2B-GFP-Cre/MYFP-H2B-Cherry-FlpO等位基因的示意图，显示了相互排斥的FRT位点对之间的遗传距离和FlpO/FlpO-ERT2重组后的预期结果。

c, d Confocal microscopy and FACS analysis of mouse ES cells used to generate Rosa26-iFlpMTomato-Cre/MYFP mice 3 days after transfection with FlpO-expressing plasmids. e-g, Frequency of recombination and expression detected by microscopy in ES cells and ECs derived from embryoid bodies (EBs). h, Relative frequency of MTomato-2A-Cre+ and MYFP+ cells in ES cells (in vitro) and in blood (in vivo); the absence of change over time shows that permanent expression of Cre is non-toxic to cell.

c、 d共聚焦显微镜和FACS分析小鼠ES细胞，用于在用表达FlpO的质粒转染后3天产生Rosa26-Ifpmtomato-Cre/MYFP小鼠。e-g，通过显微镜在源自胚状体（EB）的ES细胞和EC中检测到的重组和表达频率。h、 ES细胞（体外）和血液（体内）中MTomato-2A-Cre+和MYFP+细胞的相对频率；随着时间的推移没有变化表明Cre的永久表达对细胞无毒。

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Reprints and permissionsAbout this articleCite this articleGarcia-Gonzalez, I., Gambera, S., Rocha, S.F. et al. iFlpMosaics enable the multispectral barcoding and high-throughput comparative analysis of mutant and wild-type cells.

转载和许可本文引用本文Garcia-Gonzalez，I.，Gambera，S.，Rocha，S.F。等人。iFlpMosaics能够对突变型和野生型细胞进行多光谱条形码和高通量比较分析。

Nat Methods (2024). https://doi.org/10.1038/s41592-024-02534-wDownload citationReceived: 05 April 2024Accepted: 15 October 2024Published: 13 December 2024DOI: https://doi.org/10.1038/s41592-024-02534-wShare this articleAnyone you share the following link with will be able to read this content:Get shareable linkSorry, a shareable link is not currently available for this article.Copy to clipboard.

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