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1.  Novel ROSA26 Cre-reporter Knock-in C57BL/6N Mice Exhibiting Green Emission before and Red Emission after Cre-mediated Recombination 
Experimental Animals  2013;62(4):295-304.
The Cre/loxP system is a strategy for controlling temporal and/or spatial gene expression through genome alteration in mice. As successful Cre/loxP genome alteration depends on Cre-driver mice, Cre-reporter mice are essential for validation of Cre gene expression in vivo. In most Cre-reporter mouse strains, although the presence of reporter product indicates the expression of Cre recombinase, it has remained unclear whether a lack of reporter signal indicates either no Cre recombinase expression or insufficient reporter gene promoter activity. We produced a novel ROSA26 knock-in Cre-reporter C57BL/6N strain exhibiting green emission before and red after Cre-mediated recombination, designated as strain R26GRR. Ubiquitous green fluorescence and no red fluorescence were observed in R26GRR mice. To investigate the activation of tdsRed, EGFP-excised R26GRR, R26RR, mice were produced through the crossing of C57BL/6N mice with R26GRR/Ayu1-Cre F1 mice. R26RR mice showed extraordinarily strong red fluorescence in almost all tissues examined, suggesting ubiquitous activation of the second reporter in all tissues after Cre/loxP recombination. Moreover, endothelial cell lineage and pancreatic islet-specific expression of red fluorescence were detected in R26GRR/Tie2-Cre F1 mice and R26GRR /Ins1-Cre F1 mice, respectively. These results indicated that R26GRR mice are a useful novel Cre-reporter mouse strain. In addition, R26GRR mice with a pure C57BL/6N background represent a valuable source of green-to-red photoconvertible cells following Cre/loxP recombination for application in transplantation studies. The R26GRR mouse strain will be available from RIKEN BioResource Center (http://www.brc.riken.jp/lab/animal/en/).
doi:10.1538/expanim.62.295
PMCID: PMC4160954  PMID: 24172193
CAG promoter; Cre-reporter mouse; EGFP; Rosa26; tdsRed
2.  A Vector–Host System to Fingerprint Virus Tropism 
Human Gene Therapy  2012;23(10):1116-1126.
Abstract
Reporter genes are important tools for assessing vector pharmacology in vivo. Although useful, current systems are limited by (1) the need to generate a new vector for each different reporter, (2) the inability to package reporter genes in small vectors, and (3) variations in reporter gene feedback due to variations in cell-to-cell vector copy number. To circumvent these problems, we have used Cre recombinase as a “cat's paw” to activate reporter genes embedded in transgenic mice. The small Cre gene was introduced into self-complementary adeno-associated viral (scAAV) vectors with limited packaging capacity. Injection of scAAV-Cre vectors into mice with loxP-inactivated luciferase enabled in vivo imaging distributions comparable to the signal observed after AAV-luciferase injection. When injected into mT/mG mice, AAV-Cre converted ubiquitous expression of red fluorescent protein (RFP) to green fluorescent protein (GFP) expression only where the vectors transduced cells. Injection into F1 hybrid luciferase and mT/mG mice enabled simultaneous three-reporter tracking. This system was able to discriminate cell-specific transduction in all organs tested, with particular usefulness for detecting AAV serotype-specific transduction in the liver, kidney, and muscle. Given that F1 mice bear exactly one copy of luciferase and one copy of RFP-GFP, each reporter gene is either “on” or “off” in a cell. The Cre system therefore provides a unique quantum method to quantify vector delivery that can be applied when vector capacity is limited.
Hillestad and colleagues use self-complementary adeno-associated viral vectors encoding the Cre recombinase gene (scAAV-Cre) to activate reporter genes embedded in transgenic Cre reporter mouse strains. scAAV-Cre injected into mice containing loxP-inactivated luciferase resulted in luciferase expression similar to AAV-Luciferase vectors. In mice in which Cre-mediated recombination converts ubiquitous red fluorescent protein (RFP) expression to green fluorescent protein (GFP) expression, GFP was detected only in scAAV-Cre-transduced cells. Hybrid mice of these strains enabled simultaneous cell-specific three-reporter tracking.
doi:10.1089/hum.2011.116
PMCID: PMC3472556  PMID: 22834781
3.  A pink mouse reports the switch from red to green fluorescence upon Cre-mediated recombination 
BMC Research Notes  2012;5:296.
Background
Targeted genetic modification in the mouse becomes increasingly important in biomedical and basic science. This goal is most often achieved by use of the Cre/loxP system and numerous Cre-driver mouse lines are currently generated. Their initial characterization requires reporter mouse lines to study the in vivo spatiotemporal activity of Cre.
Findings
Here, we report a dual fluorescence reporter mouse line, which switches expression from the red fluorescent protein mCherry to eGFP after Cre-mediated recombination. Both fluorescent proteins are expressed from the ubiquitously active and strong CAGGS promoter. Among the founders, we noticed a pink mouse line, expressing high levels of the red fluorescent protein mCherry throughout the entire body. Presence of mCherry in the living animal as well as in almost all organs was clearly visible without optical equipment. Upon Cre-activity, mCherry expression was switched to eGFP, demonstrating functionality of this reporter mouse line.
Conclusions
The pink mouse presented here is an attractive novel reporter line for fluorescence-based monitoring of Cre-activity. The high expression of mCherry, which is visible to the naked eye, facilitates breeding and crossing, as no genotyping is required to identify mice carrying the reporter allele. The presence of two fluorescent proteins allows in vivo monitoring of recombined and non-recombined cells. Finally, the pink mouse is an eye-catching animal model to demonstrate the power of transgenic techniques in teaching courses.
doi:10.1186/1756-0500-5-296
PMCID: PMC3434021  PMID: 22697046
4.  Inducible Gene Manipulations in Brain Serotonergic Neurons of Transgenic Rats 
PLoS ONE  2011;6(11):e28283.
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.
doi:10.1371/journal.pone.0028283
PMCID: PMC3226688  PMID: 22140568
5.  Efficient delivery of Cre-recombinase to neurons in vivo and stable transduction of neurons using adeno-associated and lentiviral vectors 
BMC Neuroscience  2004;5:4.
Background
Inactivating genes in vivo is an important technique for establishing their function in the adult nervous system. Unfortunately, conventional knockout mice may suffer from several limitations including embryonic or perinatal lethality and the compensatory regulation of other genes. One approach to producing conditional activation or inactivation of genes involves the use of Cre recombinase to remove loxP-flanked segments of DNA. We have studied the effects of delivering Cre to the hippocampus and neocortex of adult mice by injecting replication-deficient adeno-associated virus (AAV) and lentiviral (LV) vectors into discrete regions of the forebrain.
Results
Recombinant AAV-Cre, AAV-GFP (green fluorescent protein) and LV-Cre-EGFP (enhanced GFP) were made with the transgene controlled by the cytomegalovirus promoter. Infecting 293T cells in vitro with AAV-Cre and LV-Cre-EGFP resulted in transduction of most cells as shown by GFP fluorescence and Cre immunoreactivity. Injections of submicrolitre quantities of LV-Cre-EGFP and mixtures of AAV-Cre with AAV-GFP into the neocortex and hippocampus of adult Rosa26 reporter mice resulted in strong Cre and GFP expression in the dentate gyrus and moderate to strong labelling in specific regions of the hippocampus and in the neocortex, mainly in neurons. The pattern of expression of Cre and GFP obtained with AAV and LV vectors was very similar. X-gal staining showed that Cre-mediated recombination had occurred in neurons in the same regions of the brain, starting at 3 days post-injection. No obvious toxic effects of Cre expression were detected even after four weeks post-injection.
Conclusion
AAV and LV vectors are capable of delivering Cre to neurons in discrete regions of the adult mouse brain and producing recombination.
doi:10.1186/1471-2202-5-4
PMCID: PMC343275  PMID: 15005815
6.  Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus 
Background
Several Cre reporter strains of mice have been described, in which a lacZ gene is turned on in cells expressing Cre recombinase, as well as their daughter cells, following Cre-mediated excision of a loxP-flanked transcriptional "stop" sequence. These mice are useful for cell lineage tracing experiments as well as for monitoring the expression of Cre transgenes. The green fluorescent protein (GFP) and variants such as EYFP and ECFP offer an advantage over lacZ as a reporter, in that they can be easily visualized without recourse to the vital substrates required to visualize β-gal in living tissue.
Results
In view of the general utility of targeting the ubiquitously expressed ROSA26 locus, we constructed a generic ROSA26 targeting vector. We then generated two reporter lines of mice by inserting EYFP or ECFP cDNAs into the ROSA26 locus, preceded by a loxP-flanked stop sequence. These strains were tested by crossing them with transgenic strains expressing Cre in a ubiquitous (β-actin-Cre) or a cell-specific (Isl1-Cre and En1-Cre) pattern. The resulting EYFP or ECFP expression patterns indicated that the reporter strains function as faithful monitors of Cre activity.
Conclusions
In contrast to existing lacZ reporter lines, where lacZ expression cannot easily be detected in living tissue, the EYFP and ECFP reporter strains are useful for monitoring the expression of Cre and tracing the lineage of these cells and their descendants in cultured embryos or organs. The non-overlapping emission spectra of EYFP and ECFP make them ideal for double labeling studies in living tissues.
doi:10.1186/1471-213X-1-4
PMCID: PMC31338  PMID: 11299042
7.  Conditional Bicistronic Cre Reporter Line Expressing Both Firefly Luciferase and β-galactosidase 
Molecular Imaging and Biology  2010;13(2):284-292.
Purpose
The Cre-loxP system has become an important strategy for conditional gene deletion and conditional gene expression in genetically engineered mice. To evaluate Cre recombinase expression, we generated reporter mice that permit both noninvasive imaging in living animals and either ex vivo histochemical/immunohistochemical tissue transgene expression analysis or quantitative enzyme analysis in the same animal.
Procedures
Transgenic reporter mice were generated in which a loxP-flanked enhanced green fluorescent protein (EGFP) reporter gene and STOP sequence are placed after the nearly ubiquitously expressed CAG promoter, but before a bicistronic transcriptional unit containing luciferase and β-galactosidase reporter gene coding sequences.
Results
After global deletion of the floxed STOP sequence by germ line Cre deletion, the reporter mouse expresses luciferase and β-galactosidase in all tissues examined. Tissue-specific expression of both reporter genes occurs in reporter mouse strains expressing Cre in skin (K14 keratin Cre), heart (myosin light chair Cre), or colon (Villin Cre).
Conclusion
The luc-galTg reporter mouse allows noninvasive imaging of target Cre activation both in living animals and in tissues and cells following necropsy, using loss of EGFP expression, gain of luciferase expression, and gain of β-galactosidase expression as alternatives within the same animal for qualitative analysis of Cre expression.
doi:10.1007/s11307-010-0333-x
PMCID: PMC3051074  PMID: 20495880
Cre recombinase; Transgenic mouse; Luciferase; β-galactosidase; Molecular imaging
8.  Brief expression of a GFP cre fusion gene in embryonic stem cells allows rapid retrieval of site-specific genomic deletions. 
Nucleic Acids Research  1997;25(16):3326-3331.
The Cre DNA recombinase of bacteriophage P1 has become a useful tool for precise genomic manipulation in embryonic stem (ES) cells that have been gene modified by homologous recombination. We have re-engineered the cre gene to allow ready identification of living Cre+cells by constructing a functional fusion between Cre and an enhanced green fluorescent protein from Aequorea victoria (GFPS65T). The GFP cre fusion gene product rapidly targeted the nucleus in the absence of any exogenous nuclear localization signal. Moreover, GFPCre catalyzed efficient DNA recombination in both a mouse 3T3 derivative cell line and in murine ES cells. Fluorescence- activated cell sorting (FACS) of transiently GFP cre -transfected ES cells not only allowed rapid and efficient isolation of Cre+cells after DNA transfection but also demonstrated that a burst of Cre expression is sufficient to commit cells to Cre-mediated 'pop-out' of loxP -tagged DNA from the genome. Thus, GFP cre allows rapid identification of living cells in which loxP - flanked DNA sequences are destined to be removed from the genome by Cre-mediated recombination without reliance on recombinational activation or inactivation of a marker gene at the target locus. In addition, the GFP cre fusion gene will prove useful in tracing tissue-specific Cre expression in transgenic animals, thereby facilitating the generation and analysis of conditional gene knockout mice.
PMCID: PMC146882  PMID: 9241248
9.  A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo 
Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing and adult tissue. An advance in this concept is Genetic Inducible Fate Mapping (GIFM), linking gene expression, cell fate, and cell behaviors in vivo, to create fate maps based on genetic lineage.
GIFM exploits X-CreER lines where X is a gene or set of gene regulatory elements that confers spatial expression of a modified bacteriophage protein, Cre recombinase (CreERT). CreERT contains a modified estrogen receptor ligand binding domain which renders CreERT sequestered in the cytoplasm in the absence of the drug tamoxifen. The binding of tamoxifen releases CreERT, which translocates to the nucleus and mediates recombination between DNA sequences flanked by loxP sites. In GIFM, recombination typically occurs between a loxP flanked Stop cassette preceding a reporter gene such as GFP.
Mice are bred to contain either a region- or cell type-specific CreER and a conditional reporter allele. Untreated mice will not have marking because the Stop cassette in the reporter prevents further transcription of the reporter gene. We administer tamoxifen by oral gavage to timed pregnant females, which provides temporal control of CreERT release and subsequent translocation to the nucleus removing the Stop cassette from the reporter. Following recombination, the reporter allele is constitutively and heritably expressed. This series of events marks cells such that their genetic history is indelibly recorded. The recombined reporter thus serves as a high fidelity genetic lineage tracer that, once on, is uncoupled from the gene expression initially used to drive CreERT.
We apply GIFM in mouse to study normal development and ascertain the contribution of genetic lineages to adult cell types and tissues. We also use GIFM to follow cells on mutant genetic backgrounds to better understand complex phenotypes that mimic salient features of human genetic disorders.
This video article guides researchers through experimental methods to successfully apply GIFM. We demonstrate the method using our well characterized Wnt1-CreERT;mGFP mice by administering tamoxifen at embryonic day (E)8.5 via oral gavage followed by dissection at E12.5 and analysis by epifluorescence stereomicroscopy. We also demonstrate how to micro-dissect fate mapped domains for explant preparation or FACS analysis and dissect adult fate-mapped brains for whole mount fluorescent imaging. Collectively, these procedures allow researchers to address critical questions in developmental biology and disease models.
doi:10.3791/1687
PMCID: PMC2846818  PMID: 20042997
10.  Directed Expression of Cre in Alveolar Epithelial Type 1 Cells 
Pulmonary alveolar epithelium is comprised of two morphologically and functionally distinct cell types, alveolar epithelial type (AT) I and AT2 cells. Genetically modified mice with cell-specific Cre/loxP-mediated knockouts of relevant genes in each respective cell type would be useful to help elucidate the relative contributions of AT1 versus AT2 cells to alveolar homeostasis. Cre has previously been efficiently expressed in AT2 cells in mouse lung with the surfactant protein (SP)-C promoter; however, no transgenic mouse expressing Cre in AT1 cells has so far been available. To develop an AT1 cell–specific transgenic Cre mouse, we generated a knockin of a Cre-IRES-DsRed cassette into exon 1 of the endogenous aquaporin 5 (Aqp5) gene, a gene expressed specifically in AT1 cells in the distal lung epithelium, resulting in the mouse line, Aqp5-Cre-IRES-DsRed (ACID). Endogenous Aqp5 and transgenic Cre in ACID mice showed a very similar pattern of tissue distribution by RT-PCR. To analyze Cre activity, ACID was crossed to two Cre reporter strains, R26LacZ and mT/mG. Double-transgenic offspring demonstrated reporter gene expression in a very high fraction of AT1 cells in the distal lung, whereas AT2 cells were negative. As expected, variable reporter expression was detected in several other tissues where endogenous Aqp5 is expressed (e.g., submandibular salivary gland and stomach). ACID mice should be of major utility in analyzing the functional contribution of AT1 cells to alveolar epithelial properties in vivo with Cre/loxP-mediated gene deletion technology.
doi:10.1165/rcmb.2009-0226OC
PMCID: PMC2937230  PMID: 19767448
loxP; aquaporin 5; lung; alveolar epithelium; reporter
11.  G2R Cre Reporter Transgenic Zebrafish 
The Cre/loxP site-specific recombination system has been widely used to manipulate DNA in vivo and to study gene function in the mouse by inducible transgenic and conditional gene targeting. To fully use this powerful genetic tool in a relatively new animal model, zebrafish, we generated reporter transgenic lines for easy detection of Cre recombinase activity in vivo. The transgenic fish lines, designated G2R, express two fluorescent protein genes, GFP and RFP, under the control of the ubiquitous promoter of the Xenopus EF1 alpha gene. The G2R animals change their color from green to red (G2R) after Cre-mediated recombination and are useful for development of cell type specific Cre transgenic lines and for cell lineage and fate mapping studies in zebrafish.
doi:10.1002/dvdy.21673
PMCID: PMC2665268  PMID: 18729206
Cre/loxP; transgenic zebrafish; Cre-reporter fish; conditional gene expression
12.  Generation and Characterization of Ins1-cre-driver C57BL/6N for Exclusive Pancreatic Beta Cell-specific Cre-loxP Recombination 
Experimental Animals  2014;63(2):183-191.
Cre/loxP system-mediated site-specific recombination is utilized to study gene function in vivo. Successful conditional knockout of genes of interest is dependent on the availability of Cre-driver mice. We produced and characterized pancreatic β cell-specific Cre-driver mice for use in diabetes mellitus research. The gene encoding Cre was inserted into the second exon of mouse Ins1 in a bacterial artificial chromosome (BAC). Five founder mice were produced by microinjection of linearized BAC Ins1-cre. The transgene was integrated between Mafa and the telomere on chromosome 15 in one of the founders, BAC Ins1-cre25. To investigate Cre-loxP recombination, BAC Ins1-cre25 males were crossed with two different Cre-reporters, R26R and R26GRR females. On gross observation, reporter signal after Cre-loxP recombination was detected exclusively in the adult pancreatic islets in both F1 mice. Immunohistological analysis indicated that Cre-loxP recombination-mediated reporter signal was colocalized with insulin in pancreatic islet cells of both F1 mice, but not with glucagon. Moreover, Cre-loxP recombination signal was already observed in the pancreatic islets at E13.5 in both F1 fetuses. Finally, we investigated ectopic Cre-loxP recombination for Ins1, because the ortholog Ins2 is also expressed in the brain, in addition to the pancreas. However, there was no Cre-loxP recombination-mediated reporter signal in the brain of both F1 mice. Our data suggest that BAC Ins1-cre25 mice are a useful Cre-driver C57BL/6N for pancreatic β cell-specific Cre-loxP recombination, except for crossing with knock-in mice carrying floxed gene on chromosome 15.
doi:10.1538/expanim.63.183
PMCID: PMC4160984  PMID: 24770644
cre-driver mice; cre-loxP recombination; diabetes; insulin1; pancreatic β cells
13.  Non-parallel recombination limits Cre-LoxP-based reporters as precise indicators of conditional genetic manipulation 
Genesis (New York, N.Y. : 2000)  2013;51(6):436-442.
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 common 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.
doi:10.1002/dvg.22384
PMCID: PMC3696028  PMID: 23441020
Mosaic analysis; lineage tracing; cell autonomous; Cre detection; non-parallel recombination
14.  A Modified RMCE-Compatible Rosa26 Locus for the Expression of Transgenes from Exogenous Promoters 
PLoS ONE  2012;7(1):e30011.
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.
doi:10.1371/journal.pone.0030011
PMCID: PMC3258265  PMID: 22253858
15.  Promiscuous recombination of LoxP alleles during gametogenesis in cornea Cre driver mice 
Molecular Vision  2008;14:562-571.
Purpose
To examine whether promiscuous Cre/LoxP recombination happens during gametogenesis in double transgenic mice carrying LoxP modified alleles and Cre transgene driven by tissue-specific promoter outside the gonads of adult mice.
Methods
Cre driver mice were crossbred with reporter mouse lines (e.g., ZEG and Rosa26R) to obtain Cre/ZEG and Cre/Rosa26R double transgenic mice. The frequency of promiscuous LoxP/Cre recombination was determined by the expression of second reporter genes in the offspring of double transgenic mice.
Results
The frequency of promiscuous LoxP/Cre recombination varied in different lines of Cre driver mice and in the sex of the same driver mice with higher penetrance in male than in female double transgenic mice. Polymerase chain reaction (PCR) and recombination analysis demonstrate that the recombination of floxed allele occurs during the transition from spermatogonia (diploid) to primary spermatocyte (tetraploid) in the testis. Thereby, target-floxed allele(s) may be ubiquitously ablated in experimental animals intended for tissue-specific gene deletion.
Conclusions
Gametogenesis-associated recombination should always be examined in tissue-specific gene ablation studies.
PMCID: PMC2274927  PMID: 18385792
16.  Detection and Organ-Specific Ablation of Neuroendocrine Cells by Synaptophysin Locus-Based BAC Cassette in Transgenic Mice 
PLoS ONE  2013;8(4):e60905.
The role of cells of the diffuse neuroendocrine system in development and maintenance of individual organs and tissues remains poorly understood. Here we identify a regulatory region sufficient for accurate in vivo expression of synaptophysin (SYP), a common marker of neuroendocrine differentiation, and report generation of Tg(Syp-EGFPloxP-DTA)147Ayn (SypELDTA) mice suitable for flexible organ-specific ablation of neuroendocrine cells. These mice express EGFP and diphtheria toxin fragment A (DTA) in SYP positive cells before and after Cre-loxP mediated recombination, respectively. As a proof of principle, we have crossed SypELDTA mice with EIIA-Cre and PB-Cre4 mice. EIIA-Cre mice express Cre recombinase in a broad range of tissues, while PB-Cre4 mice specifically express Cre recombinase in the prostate epithelium. Double transgenic EIIA-Cre; SypELDTA embryos exhibited massive cell death in SYP positive cells. At the same time, PB-Cre4; SypELDTA mice showed a substantial decrease in the number of neuroendocrine cells and associated prostate hypotrophy. As no increase in cell death and/or Cre-loxP mediated recombination was observed in non-neuroendocrine epithelium cells, these results suggest that neuroendocrine cells play an important role in prostate development. High cell type specificity of Syp locus-based cassette and versatility of generated mouse model should assure applicability of these resources to studies of neuroendocrine cell functions in various tissues and organs.
doi:10.1371/journal.pone.0060905
PMCID: PMC3632533  PMID: 23630575
17.  Conditional Gene Targeting in Mouse Pancreatic β-Cells 
Diabetes  2010;59(12):3090-3098.
OBJECTIVE
Conditional gene targeting has been extensively used for in vivo analysis of gene function in β-cell biology. The objective of this study was to examine whether mouse transgenic Cre lines, used to mediate β-cell– or pancreas-specific recombination, also drive Cre expression in the brain.
RESEARCH DESIGN AND METHODS
Transgenic Cre lines driven by Ins1, Ins2, and Pdx1 promoters were bred to R26R reporter strains. Cre activity was assessed by β-galactosidase or yellow fluorescent protein expression in the pancreas and the brain. Endogenous Pdx1 gene expression was monitored using Pdx1tm1Cvw lacZ knock-in mice. Cre expression in β-cells and co-localization of Cre activity with orexin-expressing and leptin-responsive neurons within the brain was assessed by immunohistochemistry.
RESULTS
All transgenic Cre lines examined that used the Ins2 promoter to drive Cre expression showed widespread Cre activity in the brain, whereas Cre lines that used Pdx1 promoter fragments showed more restricted Cre activity primarily within the hypothalamus. Immunohistochemical analysis of the hypothalamus from Tg(Pdx1-cre)89.1Dam mice revealed Cre activity in neurons expressing orexin and in neurons activated by leptin. Tg(Ins1-Cre/ERT)1Lphi mice were the only line that lacked Cre activity in the brain.
CONCLUSIONS
Cre-mediated gene manipulation using transgenic lines that express Cre under the control of the Ins2 and Pdx1 promoters are likely to alter gene expression in nutrient-sensing neurons. Therefore, data arising from the use of these transgenic Cre lines must be interpreted carefully to assess whether the resultant phenotype is solely attributable to alterations in the islet β-cells.
doi:10.2337/db10-0624
PMCID: PMC2992770  PMID: 20802254
18.  Controlled Cre/loxP Site-Specific Recombination in the Developing Brain in Medaka Fish, Oryzias latipes 
PLoS ONE  2013;8(6):e66597.
Background
Genetic mosaic techniques have been used to visualize and/or genetically modify a neuronal subpopulation within complex neural circuits in various animals. Neural populations available for mosaic analysis, however, are limited in the vertebrate brain.
Methodology/Principal Findings
To establish methodology to genetically manipulate neural circuits in medaka, we first created two transgenic (Tg) medaka lines, Tg (HSP:Cre) and Tg (HuC:loxP-DsRed-loxP-GFP). We confirmed medaka HuC promoter-derived expression of the reporter gene in juvenile medaka whole brain, and in neuronal precursor cells in the adult brain. We then demonstrated that stochastic recombination can be induced by micro-injection of Cre mRNA into Tg (HuC:loxP-DsRed-loxP-GFP) embryos at the 1-cell stage, which allowed us to visualize some subpopulations of GFP-positive cells in compartmentalized regions of the telencephalon in the adult medaka brain. This finding suggested that the distribution of clonally-related cells derived from single or a few progenitor cells was restricted to a compartmentalized region. Heat treatment of Tg(HSP:Cre x HuC:loxP-DsRed-loxP-GFP) embryos (0–1 day post fertilization [dpf]) in a thermalcycler (39°C) led to Cre/loxP recombination in the whole brain. The recombination efficiency was notably low when using 2–3 dpf embyos compared with 0–1 dpf embryos, indicating the possibility of stage-dependent sensitivity of heat-inducible recombination. Finally, using an infrared laser-evoked gene operator (IR-LEGO) system, heat shock induced in a micro area in the developing brains led to visualization of clonally-related cells in both juvenile and adult medaka fish.
Conclusions/Significance
We established a noninvasive method to control Cre/loxP site-specific recombination in the developing nervous system in medaka fish. This method will broaden the neural population available for mosaic analyses and allow for lineage tracing of the vertebrate nervous system in both juvenile and adult stages.
doi:10.1371/journal.pone.0066597
PMCID: PMC3692484  PMID: 23825546
19.  Phosphorylation of Mouse Immunity-Related GTPase (IRG) Resistance Proteins Is an Evasion Strategy for Virulent Toxoplasma gondii 
PLoS Biology  2010;8(12):e1000576.
GTPases of the mouse IRG protein family, mediators of resistance against Toxoplasma gondii in the mouse, are inactivated by a polymorphic kinase of the parasite, resulting in enhanced parasite virulence.
Virulence of complex pathogens in mammals is generally determined by multiple components of the pathogen interacting with the functional complexity and multiple layering of the mammalian immune system. It is most unusual for the resistance of a mammalian host to be overcome by the defeat of a single defence mechanism. In this study we uncover and analyse just such a case at the molecular level, involving the widespread intracellular protozoan pathogen Toxoplasma gondii and one of its most important natural hosts, the house mouse (Mus musculus). Natural polymorphism in virulence of Eurasian T. gondii strains for mice has been correlated in genetic screens with the expression of polymorphic rhoptry kinases (ROP kinases) secreted into the host cell during infection. We show that the molecular targets of the virulent allelic form of ROP18 kinase are members of a family of cellular GTPases, the interferon-inducible IRG (immunity-related GTPase) proteins, known from earlier work to be essential resistance factors in mice against avirulent strains of T. gondii. Virulent T. gondii strain ROP18 kinase phosphorylates several mouse IRG proteins. We show that the parasite kinase phosphorylates host Irga6 at two threonines in the nucleotide-binding domain, biochemically inactivating the GTPase and inhibiting its accumulation and action at the T. gondii parasitophorous vacuole membrane. Our analysis identifies the conformationally active switch I region of the GTP-binding site as an Achilles' heel of the IRG protein pathogen-resistance mechanism. The polymorphism of ROP18 in natural T. gondii populations indicates the existence of a dynamic, rapidly evolving ecological relationship between parasite virulence factors and host resistance factors. This system should be unusually fruitful for analysis at both ecological and molecular levels since both T. gondii and the mouse are widespread and abundant in the wild and are well-established model species with excellent analytical tools available.
Author Summary
Many pathogens manipulate the immune system of their hosts to facilitate infection and ensure transmission to subsequent hosts. The intracellular protozoan Toxoplasma gondii, a relative of the malaria parasite, is able to infect and persist in a remarkable variety of warm-blooded hosts. Indeed roughly a third of the human race carry live Toxoplasma cysts in their brains with no overt effects. Toxoplasma infection is kept at bay in many mammals (but not in humans) by a resistance system based on a family of proteins known as the immunity-related GTPase (IRG) family. IRG proteins accumulate in infected cells on the vacuoles containing the parasite and ultimately destroy them. In this paper, we show that, in the mouse, Toxoplasma can oppose the IRG system by secreting an enzyme called ROP18 into infected cells, which phosphorylates key amino acids on the IRG proteins, rendering them inactive. Not all strains of Toxoplasma can produce an active form of ROP18, but those strains that do are more virulent. We propose that individual hosts control Toxoplasma with differing efficiency, and the variation we see in ROP18 kinase activity produced by different Toxoplasma strains is an evolutionary response to this. Thus, in different mammalian hosts, each strain seeks a balance between an excess of virulence (resulting in premature death of the host) and resistance that is too efficient (resulting in clearance of the parasite and sterile immunity).
doi:10.1371/journal.pbio.1000576
PMCID: PMC3006384  PMID: 21203588
20.  In Vivo Genetic Ablation by Cre-Mediated Expression of Diphtheria Toxin Fragment A 
Genesis (New York, N.Y. : 2000)  2005;43(3):129-135.
Summary
We generated a ROSA26-eGFP-DTA mouse line by introducing an eGFP-DTA (enhanced green fluorescent protein - diphtheria toxin fragment A) cassette into the ROSA26 locus by homologous recombination in ES cells. This mouse expresses eGFP ubiquitously, but DTA expression is prevented by the presence of eGFP, a Neo cassette, and a strong transcriptional stop sequence. Mice carrying this construct are normal and fertile, indicating the absence of DTA expression. However, upon Cre-mediated excision of the floxed region DTA expression is activated, resulting in the specific ablation of Cre-expressing cells. As an example of this approach, we ablated Nkx2.5 and Wnt1-expressing cells by using the Nkx2.5-Cre and Wnt1-Cre mouse lines, respectively. We observed loss of the precise tissues in which Nkx2.5 and Wnt1 are expressed. Apart from being a general GFP reporter, the ROSA26-GFP-DTA mouse line should provide a useful resource for genetic ablation of specific groups of cells.
doi:10.1002/gene.20162
PMCID: PMC2233880  PMID: 16267821
Cre; loxP; diphtheria toxin; eGFP; Nkx2.5; Wnt1; genetic ablation; heart; midbrain
21.  A construct with fluorescent indicators for conditional expression of miRNA 
BMC Biotechnology  2008;8:77.
Background
Transgenic RNAi holds promise as a simple, low-cost, and fast method for reverse genetics in mammals. It may be particularly useful for producing animal models for hypomorphic gene function. Inducible RNAi that permits spatially and temporally controllable gene silencing in vivo will enhance the power of transgenic RNAi approach. Furthermore, because microRNA (miRNA) targeting specific genes can be expressed simultaneously with protein coding genes, incorporation of fluorescent marker proteins can simplify the screening and analysis of transgenic RNAi animals.
Results
We sought to optimally express a miRNA simultaneously with a fluorescent marker. We compared two construct designs. One expressed a red fluorescent protein (RFP) and a miRNA placed in its 3' untranslated region (UTR). The other expressed the same RFP and miRNA, but the precursor miRNA (pre-miRNA) coding sequence was placed in an intron that was inserted into the 3'-UTR. We found that the two constructs expressed comparable levels of miRNA. However, the intron-containing construct expressed a significantly higher level of RFP than the intron-less construct. Further experiments indicate that the 3'-UTR intron enhances RFP expression by its intrinsic gene-expression-enhancing activity and by eliminating the inhibitory effect of the pre-miRNA on the expression of RFP. Based on these findings, we incorporated the intron-embedded pre-miRNA design into a conditional expression construct that employed the Cre-loxP system. This construct initially expressed EGFP gene, which was flanked by loxP sites. After exposure to Cre recombinase, the transgene stopped EGFP expression and began expression of RFP and a miRNA, which silenced the expression of specific cellular genes.
Conclusion
We have designed and tested a conditional miRNA-expression construct and showed that this construct expresses both the marker genes strongly and can silence the target gene efficiently upon Cre-mediated induction of the miRNA expression. This construct can be used to increase the efficiency of making cell lines or transgenic animals that stably express miRNA targeting specific genes.
doi:10.1186/1472-6750-8-77
PMCID: PMC2569932  PMID: 18840295
22.  A Dual Infection Pseudorabies Virus Conditional Reporter Approach to Identify Projections to Collateralized Neurons in Complex Neural Circuits 
PLoS ONE  2011;6(6):e21141.
Replication and transneuronal transport of pseudorabies virus (PRV) are widely used to define the organization of neural circuits in rodent brain. Here we report a dual infection approach that highlights connections to neurons that collateralize within complex networks. The method combines Cre recombinase (Cre) expression from a PRV recombinant (PRV-267) and Cre-dependent reporter gene expression from a second infecting strain of PRV (PRV-263). PRV-267 expresses both Cre and a monomeric red fluorescent protein (mRFP) fused to viral capsid protein VP26 (VP26-mRFP) that accumulates in infected cell nuclei. PRV-263 carries a Brainbow cassette and expresses a red (dTomato) reporter that fills the cytoplasm. However, in the presence of Cre, the dTomato gene is recombined from the cassette, eliminating expression of the red reporter and liberating expression of either yellow (EYFP) or cyan (mCerulean) cytoplasmic reporters. We conducted proof-of-principle experiments using a well-characterized model in which separate injection of recombinant viruses into the left and right kidneys produces infection of neurons in the renal preautonomic network. Neurons dedicated to one kidney expressed the unique reporters characteristic of PRV-263 (cytoplasmic dTomato) or PRV-267 (nuclear VP26-mRFP). Dual infected neurons expressed VP26-mRFP and the cyan or yellow cytoplasmic reporters activated by Cre-mediated recombination of the Brainbow cassette. Differential expression of cyan or yellow reporters in neurons lacking VP26-mRFP provided a unique marker of neurons synaptically connected to dual infected neurons, a synaptic relationship that cannot be distinguished using other dual infection tracing approaches. These data demonstrate Cre-enabled conditional reporter expression in polysynaptic circuits that permits the identification of collateralized neurons and their presynaptic partners.
doi:10.1371/journal.pone.0021141
PMCID: PMC3116869  PMID: 21698154
23.  Incomplete Cre-mediated excision leads to phenotypic differences between Stra8-iCre; Mov10l1lox/lox and Stra8-iCre; Mov10l1lox/Δ mice 
Genesis (New York, N.Y. : 2000)  2013;51(7):481-490.
In the Cre-loxp system, expression level and activity of Cre recombinase in a Cre deletor line are critical because these determine not only the cell specificity of gene knockout (KO), but also the efficiency of Cre-mediated excision in a specific cell lineage. Although the spatiotemporal expression pattern of a Cre transgene is usually defined upon the generation of the mouse line, the Cre excision efficiency in a specific targeted cell lineage is rarely evaluated and often assumed to be 100%. Incomplete excision can lead to highly variable phenotypes due to mosaicism (i.e. co-existence of cells with the flox or the recombined flox allele) and this problem has long been overlooked. Here, we report that Stra8-iCre, a transgenic allele expressing codon-improved Cre recombinase (iCre) under the control of the male germ cell-specific Stra8 promoter, could efficiently delete one Mov10l1 flox allele in spermatogenic cells, whereas the excision was incomplete when two Mov10l1 flox alleles were present. The incomplete Cre-mediated excision led to a testicular phenotype that was much less severe than that in the true conditional KO (100% inactivation) mice. Our findings suggest that it is essential to determine the efficiency of Cre excision when Cre-loxp system is used for deleting genes in a specific cell lineage and the Cre; genelox / Δ genotype should be used to evaluate phenotypes instead of Cre; genelox/lox due to the fact that the latter usually bears incomplete deletion of the flox allele(s).
doi:10.1002/dvg.22389
PMCID: PMC3918465  PMID: 23554062
conditional gene knockout; Cre; loxp; piRNA; testis; germ line; phenotype; mosaicism
24.  Characterization of mice expressing Ins1 gene promoter driven CreERT recombinase for conditional gene deletion in pancreatic β-cells 
Islets  2014;6(1):e27685.
Gene manipulation using Cre-loxP recombination has proven to be an important approach for studying the impact of gene expression on pancreatic β-cell biology. We report the generation of a transgenic mouse line that enables a highly specific system for conditional gene manipulation within β-cells and achieve tissue specific and temporally regulated deletion of the Ctnnb1 (β-catenin) gene in pancreatic β-cells. cDNA encoding Cre recombinase fused to modified estrogen receptor (CreERT) under control of mouse insulin 1 gene promoter (Ins1) was used to construct the mouse line Tg(Ins1-Cre/ERT)1Lphi, also termed MIP1-CreERT. In a cross of MIP1-CreERT with a ROSA26/LacZ reporter strain, tamoxifen [Tmx] - dependent β-galactosidase expression occurred within pancreatic β-cells but not in other organ systems. Intraperitoneal glucose tolerance tests and glucose-stimulated changes in β-cell cytoplasmic calcium concentration were not adversely affected in adult MIP1-CreERT. A mouse line with floxed Ctnnb1 gene (Ctnnb1f/f) was crossed with the MIP1-CreERT line to generate a mouse model for inducible β-cell specific deletion of β-catenin gene (Ctnnb1f/f:MIP1-CreERT). Ctnnb1f/f:MIP1-CreERT mice and Ctnnb1f/f littermate controls, were injected with Tmx as adults to knock down β-catenin production in the majority of pancreatic β-cells. These mice showed normal glucose tolerance, islet cyto-architecture and insulin secretion. A novel protein fraction of 50Kd, immunoreactive with anti-β-catenin was observed in islet extracts from Ctnnb1f/f:MIP1-CreERT[Tmx] mice but not MIP1-CreERT-negative Ctnnb1f/f[Tmx] controls, indicating possible presence of a cryptic protein product of recombined Ctnnb1 gene. The MIP1-CreERT mouse line is a powerful tool for conditional manipulation of gene expression in β-cells.
doi:10.4161/isl.27685
PMCID: PMC4114654  PMID: 25483876
Cre recombinase; Ins1 gene promoter; calcium; glucose tolerance; insulin secretion; pancreatic islet; β-catenin
25.  MicroPET imaging of Cre–loxP-mediated conditional activation of a herpes simplex virus type 1 thymidine kinase reporter gene 
Gene therapy  2004;11(7):609-618.
Site-specific recombination tools such as the Cre–loxP system are used to create animal models where conditional gene deletion/activation studies are required. In the current proof of principle study, we have demonstrated that a PET reporter gene (PRG), the herpes simplex virus type 1 thymidine kinase (HSV1-tk), can be made to remain silent and can be activated by Cre–loxP-mediated recombination in cell culture and in living mice. An adenovirus carrying a silent HSV1-tk was tail-vein injected (1×109 PFU) in six transgenic mice that express Cre recombinase in their liver (Cre+) and in four control mice (Cre−). The liver-specific expression of the PRG in Cre+ mice was detected in the microPET following injection of the reporter probe, 9-[4-fluoro-3-(hydroxymethyl)butyl]guanine ([18F]-FHBG). The [18F]-FHBG accumulation in the liver in terms of percent-injected dose per gram of tissue was 7.72±1.13 for the Cre+ mice and 0.10±0.02 for the Cre− mice (P<0.05) 48 h after adenoviral injection. These results were further validated by quantitative RT-PCR, western blotting and by in vitro assays for herpes simplex virus type 1 thymidine kinase enzyme activity. Thus by using the Cre–loxP system it is possible to modulate a PRG and noninvasively monitor the extent of Cre–loxP-mediated gene activation by imaging in a microPET scanner.
doi:10.1038/sj.gt.3302194
PMCID: PMC4141556  PMID: 14724687
Cre recombinase; Cre–loxP system; positron emission tomography reporter gene; herpes simplex virus type 1 thymidine kinase; noninvasive imaging; conditional gene activation/deletion

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