Spontaneous ectopic recombination in cell-type-specific Cre mice removes loxP-flanked marker cassettes in vivo

Conditional gene targeting using the Cre/loxP technology generally includes integration of a selection marker cassette flanked by loxP recognition sites (floxed) in the target gene locus. Subsequent marker removal avoids possible impairment of gene expression or mosaicism due to partial and total deletions after Cre-mediated recombination in vivo. The use of deleter Cre mice for in vivo marker removal in floxed connexin43 mice revealed considerable mosaicism, but no selective marker removal. In addition, we noted that several Cre transgenic lines displayed spontaneous ectopic activity, reminiscent of deleter Cre mice, and required the confirmation of cell type-specific deletion in every individual mouse. When we used myosin heavy chain promoter Cre (alphaMyHC-Cre) mice for cardiomyocyte specific deletion, we observed, in addition to cardiomyocyte-restricted or complete excision, selective marker removal in a subgroup of mice as well. Thus, selective marker removal can be achieved as a byproduct of cell-type restricted deletion.     

Inducible gene manipulations in brain serotonergic neurons of transgenic rats

The serotonergic (5-HT) system has been implicated in various physiological processes and neuropsychiatric disorders, but in many aspects its role in normal and pathologic brain function is still unclear. One reason for this might be the lack of appropriate animal models which can address the complexity of physiological and pathophysiological 5-HT functioning. In this respect, rats offer many advantages over mice as they have been the animal of choice for sophisticated neurophysiological and behavioral studies. However, only recently technologies for the targeted and tissue specific modification of rat genes - a prerequisite for a detailed study of the 5-HT system - have been successfully developed. Here, we describe a rat transgenic system for inducible gene manipulations in 5-HT neurons. We generated a Cre driver line consisting of a tamoxifen-inducible CreERT2 recombinase under the control of mouse Tph2 regulatory sequences. Tissue-specific serotonergic Cre recombinase expression was detected in four transgenic TPH2-CreERT2 rat founder lines. For functional analysis of Cre-mediated recombination, we used a rat Cre reporter line (CAG-loxP.EGFP), in which EGFP is expressed after Cre-mediated removal of a loxP-flanked lacZ STOP cassette. We show an in-depth characterisation of this rat Cre reporter line and demonstrate its applicability for monitoring Cre-mediated recombination in all major neuronal subpopulations of the rat brain. Upon tamoxifen induction, double transgenic TPH2-CreERT2/CAG-loxP.EGFP rats show selective and efficient EGFP expression in 5-HT neurons. Without tamoxifen administration, EGFP is only expressed in few 5-HT neurons which confirms minimal background recombination. This 5-HT neuron specific CreERT2 line allows Cre-mediated, inducible gene deletion or gene overexpression in transgenic rats which provides new opportunities to decipher the complex functions of the mammalian serotonergic system.     

Strong and ubiquitous expression of transgenes targeted into the beta-actin locus by Cre/lox cassette replacement

Conventional approaches to produce transgenic mice recurrently yield unpredictable patterns and levels of transgene expression, a situation calling for the development of new techniques to overcome these drawbacks in the context of overexpression studies. Here we present an efficient method for rapid and reproducible transgenesis using the recombinase mediated cassette exchange (RMCE) (Bouhassira et al.: Blood 90:3332-3344, 1997) procedure. A lox511-EGFP-TK/neo-loxP cassette was placed under the control of the endogenous mouse beta-actin promoter. Heterozygous mice revealed strong and ubiquitous EGFP expression throughout embryogenesis and adulthood. Reproducibly, the same expression pattern was obtained with RMCE when it was used to replace the EGFP-harboring cassette by ECFP or placental alkaline phosphatase (PLAP) reporter genes (DePrimo et al.: Transgenic Res 5:459-466, 1996). Furthermore, the RMCE procedure proved efficient as well in embryonic stem (ES) cells as directly in zygotes. Our results demonstrate ubiquitous expression of floxed transgenes in the endogenous beta-actin locus and they support the general use of the beta-actin locus for targeted transgenesis.     

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 Cre recombinase transgene with mosaic, widespread tamoxifen-inducible action

Cre-mediated site-specific recombination allows conditional transgene expression or gene knockouts in mice. Inducible Cre recombination systems have been developed to bypass initial embryonic lethal phenotypes and provide access to later embryonic or adult phenotypes. We have produced Cre transgenic mice in which excision is tamoxifen inducible and occurs in a widespread mosaic pattern. We utilized our Cre excision reporter system combined with an embryonic stem (ES) cell screen to identify ES cell clones with undetectable background Cre activity in the absence of tamoxifen but efficient excision upon addition of tamoxifen. The CreER transgenic mouse lines derived from the ES cells were tested using the Z/AP and Z/EG Cre reporter lines. Reporter gene expression indicated Cre excision was maximal in midgestation embryos by 2 days after tamoxifen administration, with an overall efficiency of 5-10% of cells with Cre excision. At 3 days after tamoxifen treatment most reporter gene expression marked groups of cells, suggesting an expansion of cells with Cre excision, and the proportion of cells with Cre excision was maintained. In adults, Cre excision was also observed with varying efficiencies in all tissues after tamoxifen treatment.     

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.     

Expression of Cre Recombinase in the developing mouse limb bud driven by a Prxl enhancer

We have used a Prx1 limb enhancer to drive expression of Cre Recombinase in transgenic mice. This regulatory element leads to Cre expression throughout the early limb bud mesenchyme and in a subset of craniofacial mesenchyme. Crossing a murine line carrying this transgene to a reporter mouse harboring a floxed Cre-reporter cassette revealed that recombinase activity is first observed in the earliest limb bud at 9.5 dpc. By early to mid bud stages at 10.5 dpc recombination is essentially complete in all mesenchymal cells in the limb. Expression of the Cre recombinase was never detected in the limb bud ectoderm. The use of Prx1-Cre mice should facilitate analysis of gene function in the developing limb.     

Transgenic mice engineered to target Cre/loxP-mediated DNA recombination into catecholaminergic neurons

To introduce restricted DNA recombination events into catecholaminergic neurons using the Cre/loxP technology, we generated transgenic mice carrying the Cre recombinase gene driven by a 9 kb rat tyrosine hydroxylase (TH) promoter. Immunohistochemistry performed on transgenic mouse brain sections revealed a high number of cells expressing Cre in areas where TH is normally expressed, including the olfactory bulb, hypothalamic and midbrain dopaminergic neurons, and the locus coeruleus. Double immunohistochemistry and immunofluorescence indicated that colocalization of TH and Cre is greater than 80%. Cre expression was also found in TH-positive amacrine neurons of the retina, chromaffin cells of the adrenal medulla, and sympathetic ganglia. We crossbred TH-Cre mice with the floxed reporter strain Z/AP and observed efficient Cre-mediated recombination in all areas expressing TH, indicating that transgenic Cre is functional. Therefore, we have generated a valuable transgenic mouse strain to induce specific mutations of "floxed" genes in catecholaminergic neurons.     

Flp recombinase transgenic mice of C57BL/6 strain for conditional gene targeting

We constructed an expression vector of Flp recombinase modified by adding a nuclear localization signal. Injection of the expression vector into fertilized eggs of the C57BL/6 strain yielded transgenic mouse lines expressing the Flp recombinase transgene in the testis. We crossed the transgenic mice to reporter mice carrying the neomycin phosphotransferase gene flanked by target sites of Flp recombinase. Examination of the deletion of the neomycin phosphotransferase gene in the progeny showed that Flp-mediated recombination took place efficiently in vivo in FLP66 transgenic mouse line. These results suggest that the Flp recombinase system is effective in mice and in combination with the Cre recombinase system extends the potentials of gene manipulation in mice. One of the useful applications of FLP66 transgenic mouse line is the removal of marker genes from mice manipulated for the conditional gene targeting with the Cre/loxP system in the pure C57BL/6 genetic background.     

Foxb1-driven Cre expression in somites and the neuroepithelium of diencephalon, brainstem, and spinal cord

We have knocked-in Cre-IRES-EGFP in the Foxb1 locus by homologous recombination in embryonic stem cells. We removed the PGK-neo cassette (which was flanked by FRT sequences) by crossing with the FLPeR deleter mouse. The Foxb1(Cre) line showed Cre recombinase activity as well as EGFP fluorescence reproducing Foxb1 expression accurately. By crossing Foxb1(Cre) mice with the ROSA26R and Z/AP mouse reporter lines we have been able to trace the lineage of Foxb1-expressing cells. Early transient expression of Foxb1 in the paraxial mesoderm translates into labeling of the somites. In the central nervous system (CNS), the Foxb1 lineage includes the thalamus and mammillary body (hypothalamus), brainstem, and the ventral spinal cord and floor plate.     

Z/EG, a double reporter mouse line that expresses enhanced green fluorescent protein upon Cre-mediated excision

The Cre/loxP system has become an important tool in designing postintegrational switch mechanisms for transgenes in mice. The power and spectrum of application of this system depends on transgenic mouse lines that provide Cre recombinase activity with a defined cell type-, tissue-, or developmental stage-specificity. We have developed a novel mouse line that acts as a Cre reporter. The mice, designated Z/EG (lacZ/EGFP), express lacZ throughout embryonic development and adult stages. Cre excision, however, removes the lacZ gene, which activates expression of the second reporter, enhanced green fluorescent protein. We have found that the double-reporter Z/EG line is able to indicate the occurrence of Cre excision from early embryonic to adult lineages. The advantage of the Z/EG line is that Cre-mediated excision can be monitored in live samples and that live cells with Cre-mediated excision can be isolated using a single-step FACS. It will be a valuable reagent for the increasing number of investigators taking advantage of the powerful tools provided by the Cre/loxP site-specific recombinase system.     

Generation of a mouse with conditionally activated signaling through the BMP receptor, ALK2

BMP signaling plays pleiotropic roles in various tissues. Transgenic mouse lines that overexpress BMP signaling in a tissue-specific manner would be beneficial; however, production of each tissue-specific transgenic mouse line is labor-intensive. Here, using a Cre-loxP system, we generated a conditionally overexpressing mouse line for BMP signaling through the type I receptor ALK2 (alternatively known as AVCRI, ActRI, or ActRIA). By mating this line with Cre-expression mouse lines, Cre-mediated recombination removes an intervening floxed lacZ expression cassette and thereby permits the expression of a constitutively active form of Alk2 (caAlk2) driven by a ubiquitous promoter, CAG. Tissue specificity of Cre recombination was monitored by a bicistronically expressed EGFP following Alk2 cDNA. Increased BMP signaling was confirmed by ectopic phosphorylation of SMAD1/5/8 in the areas where Cre recombination had occurred. The conditional overexpression system described here provides versatility in investigating gene functions in a tissue-specific manner without having to generate independent tissue-specific transgenic lines.     

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 modified RMCE-compatible Rosa26 locus for the expression of transgenes from exogenous promoters

Generation of gain-of-function transgenic mice by targeting the Rosa26 locus has been established as an alternative to classical transgenic mice produced by pronuclear microinjection. However, targeting transgenes to the endogenous Rosa26 promoter results in moderate ubiquitous expression and is not suitable for high expression levels. Therefore, we now generated a modified Rosa26 (modRosa26) locus that combines efficient targeted transgenesis using recombinase-mediated cassette exchange (RMCE) by Flipase (Flp-RMCE) or Cre recombinase (Cre-RMCE) with transgene expression from exogenous promoters. We silenced the endogenous Rosa26 promoter and characterized several ubiquitous (pCAG, EF1α and CMV) and tissue-specific (VeCad, αSMA) promoters in the modRosa26 locus in vivo. We demonstrate that the ubiquitous pCAG promoter in the modRosa26 locus now offers high transgene expression. While tissue-specific promoters were all active in their cognate tissues they additionally led to rare ectopic expression. To achieve high expression levels in a tissue-specific manner, we therefore combined Flp-RMCE for rapid ES cell targeting, the pCAG promoter for high transgene levels and Cre/LoxP conditional transgene activation using well-characterized Cre lines. Using this approach we generated a Cre/LoxP-inducible reporter mouse line with high EGFP expression levels that enables cell tracing in live cells. A second reporter line expressing luciferase permits efficient monitoring of Cre activity in live animals. Thus, targeting the modRosa26 locus by RMCE minimizes the effort required to target ES cells and generates a tool for the use exogenous promoters in combination with single-copy transgenes for predictable expression in mice.     

Labeling of hematopoietic stem and progenitor cells in novel activatable EGFP reporter mice

Conditional activation and inactivation of genes using the Cre/loxP recombination system is a powerful tool for the analysis of gene function and for tracking cell fate. Here we report a novel silent EGFP reporter mouse line generated by enhancer trap technology using embryonic stem (ES) cells. Following transfection with the silent EGFP reporter construct, positive ES cell clones were treated with Cre recombinase. These "activated clones" were then further selected on the basis of ubiquitous EGFP expression during in vitro differentiation. The parental "silent" clones were then used for generating mice. Upon Cre-mediated activation in ovo tissues tested from these mice express EGFP. Long-term, strong and sustainable expression of EGFP is observed in most myeloid and lymphoid cells. As shown by in vivo transplantation assays, the majority of hematopoietic stem cells (HSCs) and spleen colony-forming units (CFU-S) reside within the EGFP positive fraction. Most in vitro colony-forming units (CFU-Cs) isolated from bone marrow also express EGFP. Thus, these reporter mice are useful for the analysis of Cre-mediated recombination in HSCs and hematopoietic progenitor cells. This, in combination with the high accessibility of the loxP sites, makes these mice a valuable tool for testing cell/tissue-specific Cre-expressing mice. .     

Thyrocyte-specific expression of Cre recombinase in transgenic mice

A transgenic mouse line that expresses Cre recombinase under control of the human thyroid peroxidase (TPO) gene promoter was established. The activity and specificity of the TPO-driven Cre recombinase were examined by using Northern blotting and by crossing with the ROSA26 reporter transgenic mouse line. In the latter mice, Cre-mediated recombination occurred only in the thyrocytes, and recombination commenced around embryonic day 14.5, at the time during thyroid organogenesis when TPO expression begins. This study demonstrates that the TPO-Cre transgenic mouse is a powerful tool to specifically delete loxP-inserted (floxed) genes in thyrocytes and will be of great value in the study of thyrocyte-specific genes during development and/or in adult thyroids.