Non‐parallel recombination limits cre‐loxP‐based reporters as precise indicators of conditional genetic manipulation

Cre/LoxP‐mediated recombination allows for conditional gene activation or inactivation. When combined with an independent lineage‐tracing reporter allele, this technique traces the lineage of presumptive genetically modified Cre‐expressing cells. Several studies have suggested that floxed alleles have differential sensitivities to Cre‐mediated recombination, which raises concerns regarding utilization of Cre‐reporters to monitor recombination of other floxed loci of interest. Here, we directly investigate the recombination correlation, at cellular resolution, between several floxed alleles induced by Cre‐expressing mouse lines. The recombination correlation between different reporter alleles varied greatly in otherwise genetically identical cell types. The chromosomal location of floxed alleles, distance between LoxP sites, sequences flanking the LoxP sites, and the level of Cre activity per cell all likely contribute to observed variations in recombination correlation. These findings directly demonstrate that, due to non‐parallel recombination events, commonly available Cre reporter mice cannot be reliably utilized, in all cases, to trace cells that have DNA recombination in independent‐target floxed alleles, and that careful validation of recombination correlations are required for proper interpretation of studies designed to trace the lineage of genetically modified populations, especially in mosaic situations. genesis 51:436–442. © 2013 Wiley Periodicals, Inc.     

Specific and spatial labeling of P0‐Cre versus Wnt1‐Cre in cranial neural crest in early mouse embryos

P0‐Cre and Wnt1‐Cre mouse lines have been widely used in combination with loxP‐flanked mice to label and genetically modify neural crest (NC) cells and their derivatives. Wnt1‐Cre has been regarded as the gold standard and there have been concerns about the specificity of P0‐Cre because it is not clear about the timing and spatial distribution of the P0‐Cre transgene in labeling NC cells at early embryonic stages. We re‐visited P0‐Cre and Wnt1‐Cre models in the labeling of NC cells in early mouse embryos with a focus on cranial NC. We found that R26‐lacZ Cre reporter responded to Cre activity more reliably than CAAG‐lacZ Cre reporter during early embryogenesis. Cre immunosignals in P0‐Cre and reporter (lacZ and RFP ) activity in P0‐Cre/R26‐lacZ and P0‐Cre/R26‐RFP embryos was detected in the cranial NC and notochord regions in E8.0–9.5 (4–19 somites) embryos. P0‐Cre transgene expression was observed in migrating NC cells and was more extensive in the forebrain and hindbrain but not apparent in the midbrain. Differences in the Cre distribution patterns of P0‐Cre and Wnt1‐Cre were profound in the midbrain and hindbrain regions, that is, extensive in the midbrain of Wnt1‐Cre and in the hindbrain of P0‐Cre embryos. The difference between P0‐Cre and Wnt1‐Cre in labeling cranial NC may provide a better explanation of the differential distributions of their NC derivatives and of the phenotypes caused by Cre‐driven genetic modifications.     

Generation of a reporter mouse line expressing Akt and EGFP upon Cre-mediated recombination

The serine-threonine kinase Akt regulates multiple biological processes. An important strategy to study Akt signaling in different tissues is targeted activation of this pathway in vivo. The current studies describe the generation of a mouse model that combines a double reporter system with activation of a constitutively active form of Akt1 (caAkt) in a Cre-dependent manner. Before Cre recombination, these mice express LacZ during development as well as in most adult tissues. After Cre-mediated excision of the LacZ reporter, functionality of the transgene was demonstrated by expression of the caAkt mutant along with the second reporter, EGFP in different pancreatic compartments and in the nervous system. This animal model provides a critical reagent for assessing the effects of Akt activation in specific tissues. The lineage-tracing properties provide a useful tool to study the role of Akt signaling in regulation of differentiation programs during development and plasticity of mature tissues.     

A Cre/LoxP conditional luciferase reporter transgenic mouse for bioluminescence monitoring of tumorigenesis

Genetically engineered, Cre/LoxP‐conditional mouse models of cancer are designed to investigate the genetic contributors of tumorigenesis and are well suited to assess therapeutic treatment responses. The capacity to serially visualize tumor burden in a noninvasive fashion would greatly strengthen their applications. We report the generation of a bioluminescent reporter strain that allows monitoring of tumor development in preexisting conditional mouse tumor models. We demonstrate that, in a Cre‐dependent glioblastoma multiforme model, tumor initiation and progression is readily monitored over time and that luminescent output is related to tumor volume. Our results show that this reporter strain may be combined with various Cre/loxP mouse tumor models to allow for noninvasive longitudinal monitoring of tumor growth and therapeutic response in vivo. genesis 47:659–666, 2009. © 2009 Wiley‐Liss, Inc.     

A multifunctional reporter mouse line for Cre‐ and FLP‐dependent lineage analysis

The Cre/lox and FLP/FRT recombination systems have been used extensively for both conditional knockout and cell lineage analysis in mice. Here we report a new multifunctional Cre/FLP dual reporter allele (R26^NZG) that exhibits strong and apparently ubiquitous marker expression in embryos and adults. The reporter construct, which is driven by the CAG promoter, was knocked into the ROSA26 locus providing an open chromatin domain for consistent expression and avoiding site‐of‐integration effects often observed with transgenic reporters. R26^NZG directs Cre‐dependent nuclear‐localized β‐galactosidase (β‐gal) expression, and can be converted into a Cre‐dependent EGFP reporter (R26^NG) by germline excision of the FRT‐flanked nlslacZ cassette. Alternatively, germline excision of the floxed PGKNEO cassette in R26^NZG generates an FLP‐dependent EGFP reporter (R26^ZG) that expresses β‐gal in FLP‐nonexpressing cells. Finally, by the simultaneous use of both Cre and FLP deleters, R26^NZG allows lineage relationships to be interrogated with greater refinement than is possible with single recombinase reporter systems. genesis 47:107–114, 2009. © 2009 Wiley‐Liss, Inc.     

A Pdgf‐cCreERT2 knock‐in mouse model for tracing PDGF‐C cell lineages during development

PDGF‐C, a member of the platelet‐derived growth factor (PDGF) family, plays important roles in the development of craniofacial structures, the neural system, the vascular system, and tumors. PDGF‐C could also be required for the regulation of certain types of stem or progenitor cells as suggested by its expression in the regions where these cells are located. To further characterize the role of PDGF‐C in development, we generated a Pdgf‐c^CreERT2 mouse strain, in which a tamoxifen‐inducible Cre (CreERT2) cDNA was specifically targeted into the Pdgf‐c genomic locus and controlled by the endogenous Pdgf‐c regulatory elements. We also showed that Cre activity in this mouse strain could be specifically induced by tamoxifen, which allowed the fate of PDGF‐C‐expressing cells to be traced at various stages of development. Using this model system, we demonstrated for the first time that PDGF‐C‐expressing cells could be multipotent, generating multiple cell lineages required for the formation of the cerebellum. Therefore, the Pdgf‐c^CreERT2 mouse strain generated in this study will be a valuable transgenic tool for exploring the function of PDGF‐C in development and stem cell biology.     

Development of a general‐purpose method for cell purification using Cre/loxP‐mediated recombination

A mammalian body is composed of more than 200 different types of cells. The purification of a certain cell type from tissues/organs enables a wide variety of studies. One popular cell purification method is immunological isolation, using antibodies against specific cell surface antigens. However, this is not a general‐purpose method, since suitable antigens have not been found in certain cell types, including embryonic gonadal somatic cells and Sertoli cells. To address this issue, we established a knock‐in mouse line, named R26 KI, designed to express the human cell surface antigen hCD271 through Cre/loxP‐mediated recombination. First, we used the R26 Kl mouse line to purify embryonic gonadal somatic cells. Gonadal somatic cells were purified from the R26 KI; Nr5a1‐Cre‐transgenic (tg) embryos almost equally as efficiently as from Nr5a1‐hCD271‐tg embryos. Second, we used the R26 KI mouse line to purify Sertoli cells successfully from R26 KI; Amh‐Cre‐tg testes. In summary, we propose that the R26 KI mouse line is a powerful tool for the purification of various cell types. genesis 53:387–393, 2015. © 2015 Wiley Periodicals, Inc.     

Cre reporter mouse expressing a nuclear localized fusion of GFP and beta-galactosidase reveals new derivatives of Pax3-expressing precursors

A new Cre-reporter strain of mouse has been developed that expresses a fusion protein derived from the lacZ gene fused to GFP with a nuclear localization signal. This construct is expressed from the ROSA26 locus upon Cre-mediated recombination that removes a loxP-flanked PGK-neo cassette, thus allowing for detection of Cre activity in all tissues. This reporter strain, which is similar to prior R26R and R26EGFP strains, has certain advantages related to the nuclear expression and the combined expression of both beta-galactosidase and GFP activities. We show that the use of this newly developed reporter line allows for enhanced resolution, detection and co-localization. Thus, we report a previously unrecognized subset of venous endothelial cells derived from Pax3 expressing precursors.     

A novel Axin2 knock‐in mouse model for visualization and lineage tracing of WNT/CTNNB1 responsive cells

Wnt signal transduction controls tissue morphogenesis, maintenance and regeneration in all multicellular animals. In mammals, the WNT/CTNNB1 (Wnt/β‐catenin) pathway controls cell proliferation and cell fate decisions before and after birth. It plays a critical role at multiple stages of embryonic development, but also governs stem cell maintenance and homeostasis in adult tissues. However, it remains challenging to monitor endogenous WNT/CTNNB1 signaling dynamics in vivo. Here, we report the generation and characterization of a new knock‐in mouse strain that doubles as a fluorescent reporter and lineage tracing driver for WNT/CTNNB1 responsive cells. We introduced a multi‐cistronic targeting cassette at the 3′ end of the universal WNT/CTNNB1 target gene Axin2. The resulting knock‐in allele expresses a bright fluorescent reporter (3xNLS‐SGFP2) and a doxycycline‐inducible driver for lineage tracing (rtTA3). We show that the Axin2^{P2A‐rtTA3‐T2A‐3xNLS‐SGFP2} strain labels WNT/CTNNB1 responsive cells at multiple anatomical sites during different stages of embryonic and postnatal development. It faithfully reports the subtle and dynamic changes in physiological WNT/CTNNB1 signaling activity that occur in vivo. We expect this mouse strain to be a useful resource for biologists who want to track and trace the location and developmental fate of WNT/CTNNB1 responsive stem cells in different contexts.     

A novel reporter mouse strain that expresses enhanced green fluorescent protein upon Cre-mediated recombination

The success of Cre-mediated conditional gene targeting depends on the specificity of Cre recombinase expression in Cre-transgenic mouse lines. As a tool to evaluate the specificity of Cre expression, we developed a reporter transgenic mouse strain that expresses enhanced green fluorescent protein (EGFP) upon Cre-mediated recombination. We demonstrate that the progeny resulting from a cross between this reporter strain and a transgenic strain expressing Cre in zygotes show ubiquitous EGFP fluorescence. This reporter strain should be useful to monitor the Cre expression directed by various promoters in transgenic mice, including mice in which Cre is expressed transiently during embryogenesis under a developmentally regulated promoter.     

A series of Cre‐ERT2 drivers for manipulation of the skeletal muscle lineage

We report the generation of five mouse strains with the tamoxifen‐inducible Cre (Cre‐ER^T2; CE) gene cassette knocked into the endogenous loci of Pax3, Myod1, Myog, Myf6, and Myl1, collectively as a resource for the skeletal muscle research community. We characterized these CE strains using the Cre reporter mice, R26R^LacZ, during embryogenesis and show that they direct tightly controlled tamoxifen‐inducible reporter expression within the expected cell lineage determined by each myogenic gene. We also examined a few selected adult skeletal muscle groups for tamoxifen‐inducible reporter expression. None of these new CE alleles direct reporter expression in the cardiac muscle. All these alleles follow the same knock‐in strategy by replacing the first exon of each gene with the CE cassette, rendering them null alleles of the endogenous gene. Advantages and disadvantages of this design are discussed. Although we describe potential immediate use of these strains, their utility likely extends beyond foreseeable questions in skeletal muscle biology. genesis 52:759–770, 2014. © 2014 Wiley Periodicals, Inc.     

A new genetic tool to improve immune‐compromised mouse models: Derivation and CRISPR/Cas9‐mediated targeting of NRG embryonic stem cell lines

Development of human hematopoietic stem cells and differentiation of embryonic stem (ES) cells/induced pluripotent stem (iPS) cells to hematopoietic stem cells are poorly understood. NOD (Non‐obese diabetic)‐derived mouse strains, such as NSG (NOD‐Scid‐il2Rg) or NRG (NOD‐Rag1‐il2Rg), are the best available models for studying the function of fetal and adult human hematopoietic cells as well as ES/iPS cell‐derived hematopoietic stem cells. Unfortunately, engraftment of human hematopoietic stem cells is very variable in these models. Introduction of additional permissive mutations into these complex genetic backgrounds of the NRG/NSG mice by natural breeding is a very demanding task in terms of time and resources. Specifically, since the genetic elements defining the NSG/NRG phenotypes have not yet been fully characterized, intense backcrossing is required to ensure transmission of the full phenotype. Here we describe the derivation of embryonic stem cell (ESC) lines from NRG pre‐implantation embryos generated by in vitro fertilization followed by the CRISPR/CAS9 targeting of the Gata‐2 locus. After injection into morula stage embryos, cells from three tested lines gave rise to chimeric adult mice showing high contribution of the ESCs (70%–100%), assessed by coat color. Moreover, these lines have been successfully targeted using Cas9/CRISPR technology, and the mutant cells have been shown to remain germ line competent. Therefore, these new NRG ESC lines combined with genome editing nucleases bring a powerful genetic tool that facilitates the generation of new NOD‐based mouse models with the aim to improve the existing xenograft models.     

Recombinase-mediated cassette exchange (RMCE) — A rapidly-expanding toolbox for targeted genomic modifications

Starting in 1991, the advance of Tyr-recombinases Flp and Cre enabled superior strategies for the predictable insertion of transgenes into compatible target sites of mammalian cells. Early approaches suffered from the reversibility of integration routes and the fact that co-introduction of prokaryotic vector parts triggered uncontrolled heterochromatization. Shortcomings of this kind were overcome when Flp-Recombinase Mediated Cassette Exchange entered the field in 1994. RMCE enables enhanced tag-and-exchange strategies by precisely replacing a genomic target cassette by a compatible donor construct. After “gene swapping” the donor cassette is safely locked in, but can nevertheless be re-mobilized in case other compatible donor cassettes are provided (“serial RMCE”). These features considerably expand the options for systematic, stepwise genome modifications. The first decade was dominated by the systematic generation of cell lines for biotechnological purposes. Based on the reproducible expression capacity of the resulting strains, a comprehensive toolbox emerged to serve a multitude of purposes, which constitute the first part of this review. The concept per se did not, however, provide access to high-producer strains able to outcompete industrial multiple-copy cell lines. This fact gave rise to systematic improvements, among these certain accumulative site-specific integration pathways. The exceptional value of RMCE emerged after its entry into the stem cell field, where it started to contribute to the generation of induced pluripotent stem (iPS-) cells and their subsequent differentiation yielding a variety of cell types for diagnostic and therapeutic purposes. This topic firmly relies on the strategies developed in the first decade and can be seen as the major ambition of the present article. In this context an unanticipated, potent property of serial Flp-RMCE setups concerns the potential to re-open loci that have served to establish the iPS status before the site underwent the obligatory silencing process. Other relevant options relate to the introduction of composite Flp-recognition target sites (“heterospecific FRT-doublets”), into the LTRs of lentiviral vectors. These “twin sites” enhance the safety of iPS re-programming and -differentiation as they enable the subsequent quantitative excision of a transgene, leaving behind a single “FRT-twin”. Such a strategy combines the established expression potential of the common retro- and lentiviral systems with options to terminate the process at will. The remaining genomic tag serves to identify and characterize the insertion site with the goal to identify genomic “safe harbors” (GOIs) for re-use. This is enabled by the capacity of “FRT-twins” to accommodate any incoming RMCE-donor cassette with a compatible design.