A mouse model with tamoxifen‐inducible thyrocyte‐specific cre recombinase activity

We have created a mouse model expressing tamoxifen‐inducible Cre recombinase (CreER^T2) under the control of the thyroglobulin (Tg) gene promoter to be able to study the role of defined genetic modifications in the regulation of thyroid function. We chose the thyroglobulin promoter, as it is expressed specifically in the thyroid. In order to obtain reliable expression under the control of the Tg promoter, we used a P1 artificial chromosome (PAC) containing a large piece of the Tg promoter. A tamoxifen inducible CreER^T2 construct was selected to avoid the possible consequences of the gene deletion for the development of the thyroid gland, and to study the role of gene deletion in the adult thyroid. Transgenic lines (TgCreER^T2) carrying this construct were generated and analyzed by crossing the TgCreER^T2 mice with the ROSA26LacZ reporter strain. The activity and specificity of the Cre recombinase was tested by staining for β‐galactosidase activity and by immunohistochemistry using an anti‐Cre‐antibody. In the TgCreER^T2xROSA26LacZ reporter line, Cre‐mediated recombination occurred specifically in the thyrocytes only after tamoxifen administration, and no significant staining was observed in controls. The recombination efficiency was nearly complete, since almost all thyrocytes showed X‐gal staining. We could also induce the recombination in utero by giving tamoxifen to the pregnant female. In addition, mice expressing TgCreER^T2 had no obvious histological changes, hormonal alterations, or different response to growth stimuli as compared to controls. These results demonstrate that the TgCreER^T2 mouse line is a powerful tool to study temporally controlled deletion of floxed genes in the thyroid. genesis 52:333–340, 2014. © 2014 Wiley Periodicals, Inc.     

Generation of CD4CreERT2 transgenic mice to study development of peripheral CD4‐T‐cells

After thymic emigration CD4‐T‐cells continue to differentiate into multiple effector and suppressor sublineages in peripheral lymphoid organs. In vivo analysis of peripheral CD4‐T‐cell differentiation has relied on animal models with targeted gene mutations. These are expressed either constitutively or conditionally after Cre mediated recombination. Available Cre transgenic strains to specifically target T‐cells act at stages of thymocyte development that precede thymic selection. Tracing gene functions in CD4‐T‐cell development after thymic exit becomes complicated when the targeted gene is essential during thymic development. Other approaches to conditionally modify gene functions in peripheral T‐cells involve infection of in vitro activated cells with Cre expressing lenti‐, retro‐, or adenoviruses, which precludes in vivo analyses. To study molecular mechanisms of peripheral CD4‐T‐cell differentiation in vivo and in vitro we generated transgenic mice expressing a tamoxifen inducible Cre recombinase (CreER^T2) under the control of the CD4 gene promoter. We show here that in CD4CreER^T2 mice Cre is inducibly and selectively activated in CD4‐T‐cells. Tamoxifen treatment both in vivo and in vitro results in efficient recombination of loci marked by LoxP sites. Moreover, this strain shows no abnormalities related to transgene insertion. Therefore it provides a valuable tool for studying gene function during differentiation of naïve peripheral CD4‐T‐cells into effector or suppressor sub‐lineages. genesis 50:908–913, 2012. © 2012 Wiley Periodicals, Inc.     

Tamoxifen‐inducible Cre‐mediated recombination in adipocytes

To generate a mouse line which allows inducible, Cre/loxP‐dependent recombination in adipocytes, we used RedE/RedT‐mediated recombineering to insert the CreER^T2‐transgene, which encodes a fusion protein of Cre and a mutated tamoxifen‐responsive estrogen receptor, into the start codon of the adipocyte‐specific Adipoq gene. Adipoq encodes adiponectin, an adipokine specifically expressed in differentiated adipocytes. Tamoxifen treatment induced almost complete recombination in white adipose tissue of the AdipoqCreER^T2 mouse line (97%–99%), while no recombination was seen in vehicle‐treated animals. Recombination in brown adipose tissue was about 15%, whereas other organs and tissues did not undergo recombination. In addition, mice expressing CreER^T2 in adipocytes did not show any alterations of metabolic functions like glucose tolerance, lipolysis, or energy expenditure compared to control mice. Therefore the AdipoqCreER^T2 mouse line will be a valuable tool for studying the consequences of a temporally controlled deletion of floxed genes in white adipose tissue. genesis 48:618–625, 2010. © 2010 Wiley‐Liss, Inc.     

Highly B lymphocyte‐specific tamoxifen inducible transgene expression of CreERT2 by using the LC‐1 locus BAC vector

The generation of cell type specific inducible Cre transgenic mice is the most challenging and limiting part in the development of spatio‐temporally controlled knockout mouse models. Here we report the generation and characterization of a B lymphocyte‐specific tamoxifen‐inducible Cre transgenic mouse strain, LC‐1‐hCD19‐CreER^T2. We utilized the human CD19 promoter for expression of the tamoxifen‐inducible Cre recombinase (CreER^T2) gene, embedded in genomic sequences previously reported to give minimal position effects after transgenesis. Cre recombinase activity was evaluated by cross‐breeding the LC‐1‐hCD19‐CreER^T2 strain with a strain containing a floxed gene widely expressed in the hematopoietic system. Cre activity was only detected in the presence of tamoxifen and was restricted to B lymphocytes. The efficacy of recombination ranged from 27 to 61% in the hemizygous and homozygous mice, respectively. In conclusion, the LC‐1‐hCD19‐CreER^T2 strain is a powerful tool to study gene function specifically in B lymphocytes at any chosen time point in the lifecycle of the mouse. genesis 47:729–735, 2009. © 2009 Wiley‐Liss, Inc.     

Sequencing two Tyr::CreERT2 transgenic mouse lines

The Cre/loxP system is a powerful tool that has allowed the study of the effects of specific genes of interest in various biological settings. The Tyr::CreER^T2 system allows for the targeted expression and activity of the Cre enzyme in the melanocyte lineage following treatment with tamoxifen, thus providing spatial and temporal control of the expression of specific target genes. Two independent transgenic mouse models, each containing a Tyr::CreER^T2 transgene, have been generated and are widely used to study melanocyte transformation. In this study, we performed whole genome sequencing (WGS) on genomic DNA from the two Tyr::CreER^T2 mouse models and identified their sites of integration in the C57BL/6 genome. Based on these results, we designed PCR primers to accurately, and efficiently, genotype transgenic mice. Finally, we discussed some of the advantages of each transgenic mouse model.     

Generation of knock‐in mice that express nuclear enhanced green fluorescent protein and tamoxifen‐inducible Cre recombinase in the notochord from Foxa2 and T loci

The node and the notochord are important embryonic signaling centers that control embryonic pattern formation. Notochord progenitor cells present in the node and later in the posterior end of the notochord move anteriorly to generate the notochord. To understand the dynamics of cell movement during notochord development and the molecular mechanisms controlling this event, analyses of cell movements using time‐lapse imaging and conditional manipulation of gene activities are required. To achieve this goal, we generated two knock‐in mouse lines that simultaneously express nuclear enhanced green fluorescent protein (EGFP) and tamoxifen‐inducible Cre, CreER^T2, from two notochord gene loci, Foxa2 and T (Brachury). In Foxa2^{nEGFP‐CreERT2/+} and T^{nEGFP‐CreERT2/+} embryos, nuclei of the Foxa2 or T‐expressing cells, which include the node, notochord, and endoderm (Foxa2) or wide range of posterior mesoderm (T), were labeled with EGFP at intensities that can be used for live imaging. Cre activity was also induced in cells expressing Foxa2 and T 1 day after tamoxifen administration. These mice are expected to be useful tools for analyzing the mechanisms of notochord development. genesis 51:210–218, 2013. © 2013 Wiley Periodicals, Inc.     

The Tol2-mediated Gal4-UAS method for gene and enhancer trapping in zebrafish

The Gal4-UAS system provides powerful tools to analyze the function of genes and cells in vivo and has been extensively employed in Drosophila. The usefulness of this approach relies on the P element-mediated Gal4 enhancer trapping, which can efficiently generate transgenic fly lines expressing Gal4 in specific cells. Similar approaches, however, had not been developed in vertebrate systems due to the lack of an efficient transgenesis method. We have been developing transposon techniques by using the madaka fish Tol2 element. Taking advantage of its ability to generate genome-wide insertions, we developed the Gal4 gene trap and enhancer trap methods in zebrafish that enabled us to create various transgenic fish expressing Gal4 in specific cells. The Gal4-expressing cells can be visualized and manipulated in vivo by crossing the transgenic Gal4 lines with transgenic lines carrying various reporter and effector genes downstream of UAS (upstream activating sequence). Thus, the Gal4 gene trap and enhancer trap methods together with UAS lines now make detailed analyses of genes and cells in zebrafish feasible. Here, we describe the protocols to perform Gal4 gene trap and enhancer trap screens in zebrafish and their application to the studies of vertebrate neural circuits.     

Inducible site‐specific recombination in neural stem/progenitor cells

To establish a genetic tool for manipulating the neural stem/progenitor cell (NSC) lineage in a temporally controlled manner, we generated a transgenic mouse line carrying an NSC‐specific nestin promoter/enhancer expressing a fusion protein encoding Cre recombinase coupled to modified estrogen receptor ligand‐binding domain (ER^T2). In the background of the Cre reporter mouse strain Rosa26^lacZ, we show that the fusion CreER^T2 recombinase is normally silent but can be activated by the estrogen analog tamoxifen both in utero, in infancy, and in adulthood. As assayed by β‐galactosidase activity in embryonic stages, tamoxifen activates Cre recombinase exclusively in neurogenic cells and their progeny. This property persists in adult mice, but Cre activity can also be detected in granule neurons and Bergmann glia at the anterior of the cerebellum, in piriform cortex, optic nerve, and some peripheral ganglia. No obvious Cre activity was observed outside of the nervous system. Thus, the nestin regulated inducible Cre mouse line provides a powerful tool for studying the physiology and lineage of NSCs. genesis 47:122–131, 2009. © 2008 Wiley‐Liss, Inc.     

Temporally-Controlled Site-Specific Recombination in Zebrafish

Conventional use of the site-specific recombinase Cre is a powerful technology in mouse, but almost absent in other vertebrate model organisms. In zebrafish, Cre-mediated recombination efficiency was previously very low. Here we show that using transposon-mediated transgenesis, Cre is in fact highly efficient in this organism. Furthermore, temporal control of recombination can be achieved by using the ligand-inducible CreER^T2. Site-specific recombination only occurs upon administration of the drug tamoxifen (TAM) or its active metabolite, 4-hydroxy-tamoxifen (4-OHT). Cre-mediated recombination is detectable already 4 or 2 hours after administration of TAM or 4-OHT, demonstrating fast recombination kinetics. In addition, low doses of TAM allow mosaic labeling of single cells. Combined, our results show that conditional Cre/lox will be a valuable tool for both, embryonic and adult zebrafish studies. Furthermore, single copy insertion transgenesis of Cre/lox constructs suggest a strategy suitable also for other organisms.     

Transgenic mice exhibiting inducible and spontaneous Cre activities driven by a bovine keratin 5 promoter that can be used for the conditional analysis of basal epithelial cells in multiple organs

Background

Cre/loxP-mediated genetic modification is the most widely used conditional genetic approach used in the mouse. Engineered Cre and the mutated ligand-binding domain of estrogen receptor fusion recombinase (CreER^T) allow temporal control of Cre activity.

Results

In this study, we have generated two distinct transgenic mouse lines expressing CreER^T, which show 4-hydroxytamoxifen (4-OHT)-inducible and spontaneous (4-OHT-independent) Cre activities, referred to Tg(BK5-CreER ^T )I and Tg(BK5-CreER ^T )S, respectively. The transgenic construct is driven by the bovine Keratin 5 promoter, which is active in the basal epithelial lineage of stratified and pseudo-stratified epithelium across multiple organs. Despite the difference in 4-OHT dependency, the Tg(BK5-CreER ^T )I and Tg(BK5-CreER ^T )S mouse lines shared similar Cre-mediated recombination among various organs, except for unique mammary epithelial Cre activity in Tg(BK5-CreER ^T )S females.

Conclusion

These two new transgenic mouse lines for the analysis of basal epithelial function and for the genetic modification have been created allowing the identification of these cell lineages and analysis of their differentiation during embryogenesis, during perinatal development and in adult mice.     

A mouse line expressing Sall1‐driven inducible Cre recombinase in the kidney mesenchyme

Sall1 is expressed in the metanephric mesenchyme in the developing kidney, and mice deficient in Sall1 show kidney agenesis or dysgenesis. Sall1 is also expressed elsewhere, including in the limb buds, anus, heart, and central nervous system. Dominant‐negative mutations of Sall1 in mice and humans lead to developmental defects in these organs. Here, we generated a mouse line expressing tamoxifen‐inducible Cre recombinase (CreER^T2) under the control of the endogenous Sall1 promoter. Upon tamoxifen treatment, these mice showed genomic recombination in the tissues where endogenous Sall1 is expressed. When CreER^T2 mice were crossed with the floxed Sall1 allele, tamoxifen administration during gestation led to a significant decrease in Sall1 expression and small kidneys at birth, suggesting that Sall1 functions were disrupted. Furthermore, Sall1 expression in the kidney was significantly reduced by neonatal tamoxifen treatment. The Sall1CreER^T2 mouse is a valuable tool for in vivo time‐dependent and region‐specific knockout and overexpression studies. genesis 48:207–212, 2010. © 2010 Wiley‐Liss, Inc.     

A mouse tool for conditional mutagenesis in ovarian granulosa cells

Here we describe the generation of an inducible Cre transgenic line allowing conditional mutagenesis in ovarian granulosa cells. We have expressed the tamoxifen inducible CreER^T2 fusion protein from a Bacterial Artificial Chromosome (BAC) containing the regulatory elements of the hydroxysteroid (17‐beta) dehydrogenase 1 (Hsd17b1) gene. Hsd17b1‐iCreER^T2 transgenic mice express the iCreER^T2 fusion protein exclusively in ovarian granulosa cells. Recombination analysis at the genomic DNA level using mice with “floxed” Stat3 alleles showed no Cre activity in absence of tamoxifen whereas tamoxifen treatment induced Cre activity solely in the ovaries. Further characterization of Hsd17b1‐iCreER^T2 mice using a Cre reporter line demonstrated that Cre‐mediated recombination was restricted to ovarian granulosa cells. Therefore, Hsd17b1‐iCreER^T2 mice should be a useful tool to analyze the gene functions in ovarian granulosa cells. genesis 48:612–617, 2010. © 2010 Wiley‐Liss, Inc.     

TAILOR: Transgene Activation and Inactivation Using Lox and Rox in Zebrafish

The ability to achieve precisely tailored activation and inactivation of gene expression represents a critical utility for vertebrate model organisms. In this regard, Cre and other site-specific DNA recombinases have come to play a central role in achieving temporally regulated and cell type-specific genetic manipulation. In zebrafish, both Cre and Flp recombinases have been applied for inducible activation, inactivation and inversion of inserted genomic elements. Here we describe the addition of Dre, a heterospecific Cre-related site-specific recombinase, to the zebrafish genomic toolbox. Combining Dre-based recombination in zebrafish with established Cre/lox technology, we have established an effective strategy for transgene activation and inactivation using lox and rox (TAILOR). Using stable transgenic lines expressing tamoxifen-inducible CreER^T2 and RU486-inducible DrePR fusions, we demonstrate that Cre and Dre retain non-overlapping specificities for their respective lox and rox target sites in larval zebrafish, and that their combinatorial and sequential activation can achieve precisely timed transgene activation and inactivation. In addition to TAILOR, the successful application of Dre/rox technology in zebrafish will facilitate a variety of additional downstream genetic applications, including sequential lineage labeling, complex genomic rearrangements and the precise temporal and spatial control of gene expression through the intersection of partially overlapping promoter activities.     

Effects of CreERT2, 4-OH Tamoxifen,and Gender on CFU-F Assays

Gene function in stem cell maintenance is often tested by inducing deletion via the Cre-loxP system. However, controls for Cre and other variables are frequently not included. Here we show that when cultured in the presence of 4-OH tamoxifen, bone and marrow cells containing the CreER^T2 construct have a reduced colony forming ability. Inactive CreER^T2 recombinase, however, has the opposite effect. Young female marrow cells containing the inactive CreER^T2 construct grew more colonies than cells lacking the construct altogether. Young female control marrow cells (i.e., negative for CreER^T2) also produced significantly greater colony numbers when cultured with 4-OH tamoxifen, compared with the ethanol vehicle control. In conclusion, we report that the use of the Cre-loxP system is inadvisable in combination with CFU-F assays, and that appropriate controls should be in place to extend the future use of Cre-loxP in alternate assays.     

Generation of a conditional lima1a allele in zebrafish using the FLEx switch technology

Gene trapping has emerged as a valuable tool to create conditional alleles in various model organisms. Here we report the FLEx‐based gene trap vector SAGFLEx that allows the generation of conditional mutations in zebrafish by gene‐trap mutagenesis. The SAGFLEx gene‐trap cassette comprises the rabbit β‐globin splice acceptor and the coding sequence of GFP, flanked by pairs of inversely oriented heterotypic target sites for the site‐specific recombinases Cre and Flp. Insertion of the gene‐trap cassette into endogenous genes can result in conditional mutations that are stably inverted by Cre and Flp, respectively. To test the functionality of this system we performed a pilot screen and analyzed the insertion of the gene‐trap cassette into the lima1a gene locus. In this lima1a allele, GFP expression faithfully recapitulated the endogenous lima1a expression and resulted in a complete knockout of the gene in homozygosity. Application of either Cre or Flp was able to mediate the stable inversion of the gene trap cassette and showed the ability to conditionally rescue or reintroduce the gene inactivation. Combined with pharmacologically inducible site specific recombinases the SAGFLEx vector insertions will enable precise conditional knockout studies in a spatial‐ and temporal‐controlled manner. genesis 54:19–28, 2016. © 2015 Wiley Periodicals, Inc.     

Generation and characterization of a zebrafish muscle specific inducible Cre line

Zebrafish transgenic lines provide valuable insights into gene functions, cell lineages and cell behaviors during development. Spatiotemporal control over transgene expression is a critical need in many experimental approaches, with applications in loss- and gain-of-function expression, ectopic expression and lineage tracing experiments. The Cre/loxP recombination system is a powerful tool to provide this control and the demand for validated Cre and loxP zebrafish transgenics is high. One of the major challenges to widespread application of Cre/loxP technology in zebrafish is comparatively small numbers of established tissue-specific Cre or CreERT2 lines. We used Tol2-mediated transgenesis to generate Tg(CrymCherry;-1.9mylz2:CreERT2) which provides an inducible CreERT2 source driven by muscle-specific mylz2 promoter. The transgenic specifically labels the trunk and tail skeletal muscles. We assessed the temporal responsiveness of the transgenic by screening with a validated loxP reporter transgenic ubi:Switch. Further, we evaluated the recombination efficiency in the transgenic with varying concentrations of 4-OHT, for different induction time periods and at different stages of embryogenesis and observed that higher recombination efficiency is achieved when embryos are induced with 10 μM 4-OHT from 10-somites or 24 hpf till 48 or 72 hpf. The transgenic is an addition to currently available zebrafish transgenesis toolbox and a significant tool to advance muscle biology studies in zebrafish.