Simultaneous excision of two transgene flanking sequences and resolution of complex integration loci

In planta excision techniques have proven useful both for basic biology and applied biotechnology. In this report, we describe a simple site-specific recombination (SSR) strategy that simultaneously removes pre-defined DNA sequences from both sides of a transgenic "gene of interest," leaving only the desired gene and short sequences from the recombinase recognition site. We have used the FLP/FRT SSR system to provide a proof of concept, though any of several other SSR systems could be used in the same way. The frequency of double excision ranged from 33% to 83% in different transgenic lines. We show that a single SSR reaction can simultaneously carry out double excisions and resolve complex transgene loci at high frequency. The method has direct biotechnological application and provides a useful tool for basic research.     

Marker-free site-specific gene integration in rice based on the use of two recombination systems

Transgene integration mediated by heterologous site-specific recombination (SSR) systems into the dedicated genomic sites has been demonstrated in a few different plant species. This approach of plant transformation generates a precise site-specific integration (SSI) structure consisting of a single copy of the transgene construct. As a result, stable transgene expression correlated with promoter strength and gene copy number is observed among independent transgenic lines and faithfully transmitted through subsequent generations. Site-specific integration approaches use selectable marker genes, removal of which is necessary for the implementation of this approach as a biotechnology application. As SSR systems are also excellent tools for excising marker genes from transgene locus, a molecular strategy involving gene integration followed by marker excision, each mediated by a distinct recombination system, was earlier proposed. Experimental validation of this approach is the focus of this work. Using FLPe-FRT system for site-specific gene integration and heat-inducible Cre-lox for marker gene excision, marker-free SSI lines were developed in the first generation itself. More importantly, progeny derived from these lines inherited the marker-free locus, indicating efficient germinal transmission. Finally, as the transgene expression from SSI locus was not altered upon marker excision, this method is suitable for streamlining the production of marker-free SSI lines.     

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.     

Temporally controlled somatic mutagenesis in smooth muscle

Ligand-dependent site-specific recombinases are powerful tools to engineer the mouse genome in specific somatic cell types at selected times during pre- and postnatal development. Current efforts are primarily directed towards increasing the efficiency of this recombination system in mice. We have generated transgenic mouse lines expressing a tamoxifen-activated Cre recombinase, CreER(T2), under the control of the smooth muscle-specific SM22 promoter. Both a randomly integrated transgene [SM-CreER(T2)(tg)] and a transgene that has been "knocked in" into the endogenous SM22 locus [SM-CreER(T2)(ki)] were expressed in smooth muscle-containing tissues. The level of CreER(T2) expression and tamoxifen-induced recombination was lower in SM-CreER(T2)(tg) mice compared with SM-CreER(T2)(ki) mice. Whereas no recombinase activity could be detected in vehicle-treated SM-CreER(T2)(ki) mice, administration of tamoxifen induced the excision of a loxP-flanked reporter transgene in up to 100% of smooth muscle cells. The recombined genome persisted for at least four months after tamoxifen treatment. SM-CreER(T2)(ki) transgenic mice should be useful to study the effects of various somatic mutations in smooth muscle.     

Reduction of Cre recombinase toxicity in proliferating Drosophila cells by estrogen-dependent activity regulation

The Cre/loxP site-specific recombination system has been used successfully for genome manipulation in a wide range of species. However, in Drosophila melanogaster, a major model organism for genetic analyses, the alternative FLP/FRT system, which is less efficient at least in mammalian cells, has been established, primarily for the generation of genetic mosaics for clonal analyses. To extend genetic methodology in D. melanogaster, we have created transgenic lines allowing tissue-specific expression of Cre recombinase with the UAS/GAL4 system. Surprisingly, chronic expression of Cre recombinase from these transgenes (UAST-cre) was found to be toxic for proliferating cells. Therefore, we also generated transgenic lines allowing the expression of Cre recombinase fused to the ligand-binding domain of the human estrogen receptor (UASP-cre-EBD). We demonstrate that recombination can be efficiently dissociated from toxicity by estrogen-dependent regulation of recombinase activity of the UASP-cre-EBD transgene products.     

FLP recombinase-mediated site-specific recombination in silkworm, Bombyx mori

A comprehensive understanding of gene function and the production of site-specific genetically modified mutants are two major goals of genetic engineering in the post-genomic era. Although site-specific recombination systems have been powerful tools for genome manipulation of many organisms, they have not yet been established for use in the manipulation of the silkworm Bombyx mori genome. In this study, we achieved site-specific excision of a target gene at predefined chromosomal sites in the silkworm using a FLP/FRT site-specific recombination system. We first constructed two stable transgenic target silkworm strains that both contain a single copy of the transgene construct comprising a target gene expression cassette flanked by FRT sites. Using pre-blastoderm microinjection of a FLP recombinase helper expression vector, 32 G3 site-specific recombinant transgenic individuals were isolated from five of 143 broods. The average frequency of FLP recombinase-mediated site-specific excision in the two target strains genome was approximately 3.5%. This study shows that it is feasible to achieve site-specific recombination in silkworms using the FLP/FRT system. We conclude that the FLP/FRT system is a useful tool for genome manipulation in the silkworm. Furthermore, this is the first reported use of the FLP/FRT system for the genetic manipulation of a lepidopteran genome and thus provides a useful reference for the establishment of genome manipulation technologies in other lepidopteran species.     

Site- and time-specific gene targeting in the mouse

The efficient introduction of somatic mutations in a given gene, at a given time, in a specific cell type, will facilitate studies of gene function and the generation of animal models for human diseases. We have established a conditional site-specific recombination system in mice using a new version of the Cre/lox system. The Cre recombinase has been fused to a mutated ligand binding domain of the human estrogen receptor (ER), resulting in a tamoxifen-dependent Cre recombinase, Cre-ER(T), that is activated by tamoxifen, but not by estradiol. Transgenic mice were generated expressing Cre-ER(T) under the control of a cytomegalovirus promoter. Administration of tamoxifen to these transgenic mice induced excision of a chromosomally integrated gene flanked by loxP sites in a number of tissues, whereas no excision could be detected in untreated animals. However, the efficiency of excision varied between tissues, and the highest level (approximately 40%) was obtained in the skin. To determine the efficiency of excision mediated by Cre-ER(T) in a given cell type, Cre-ER(T)-expressing mice were crossed with reporter mice in which expression of Escherichia coli beta-galactosidase can be induced through Cre-mediated recombination. The efficiency and kinetics of this recombination were analyzed at the cellular level in the epidermis of 6- to 8-week-old double transgenic mice. Site-specific excision occurred within a few days of tamoxifen treatment in essentially all epidermis cells expressing Cre-ER(T). These results indicate that cell-specific expression of Cre-ER(T) in transgenic mice can be used for efficient tamoxifen-dependent Cre-mediated recombination at loci containing loxP sites, to generate site-specific somatic mutations in a spatiotemporally controlled manner. This conditional site-specific recombination system should allow the analysis of knockout phenotypes that cannot be addressed by conventional gene targeting.     

Effective production of marker-free transgenic strawberry plants using inducible site-specific recombination and a bifunctional selectable marker gene

Public concerns about the issue of the environmental safety of genetically modified plants have led to a demand for technologies allowing the production of transgenic plants without selectable (antibiotic resistance) markers. We describe the development of an effective transformation system for generating such marker-free transgenic plants, without the need for repeated transformation or sexual crossing. This system combines an inducible site-specific recombinase for the precise elimination of undesired, introduced DNA sequences with a bifunctional selectable marker gene used for the initial positive selection of transgenic tissue and subsequent negative selection for fully marker-free plants. The described system can be generally applied to existing transformation protocols, and was tested in strawberry using a model vector in which site-specific recombination leads to a functional combination of a cauliflower mosaic virus 35S promoter and a GUS encoding sequence, thereby enabling the histochemical monitoring of recombination events. Fully marker-free transgenic strawberry plants were obtained following two different selection/regeneration strategies.     

Selective expression of the Cre recombinase in late-stage thymocytes using the distal promoter of the Lck gene

Transgenic mouse lines were generated that express the Cre recombinase under the control of the distal promoter of the mouse Lck gene. Cre recombination in four of these lines of transgenic mice was characterized at the single cell level using ROSA26-regulated loxP-Stop-loxP-betageo and loxP-Stop-loxP-YFP reporter mouse lines. Two of the lines showed T cell-restricted Cre recombination, whereas the other two also expressed Cre in B cells, NK cells, and monocytes. Cre recombination began at a late stage of T cell development (at or after up-regulation of the TCR during positive selection) in the two T cell-restricted lines. Lines of mice that express the Cre recombinase at late stages of thymocyte development are of value for determining the impact of mutations on T cell function in the absence of complicating effects on early thymocyte selection.     

A transformation vector for the production of marker-free transgenic plants containing a single copy transgene at high frequency

We represent here the GST-MAT vector system. The R recombinase gene of the site-specific recombination system R/RS from Zygosaccharomyces rouxii was fused to the chemical inducible promoter of the glutathione-S-transferase (GST-II-27) gene from Zea mays. Upon excision, the isopentenyltransferase (ipt) gene that is used as a selectable marker gene is removed. When the cauliflower mosaic virus 35S promoter (CaMV 35S) was used to express R recombinase, 67% of the marker-free transgenic plants had more than three transgene copies. Because the CaMV 35S promoter transiently and efficiently excised the ipt gene before callus and adventitious bud formation, the frequency of emergence of the ipt-shooty explants with a single T-DNA copy might be reduced. In this study we show that the GST-MAT vector efficiently produced transgenic ipt-shooty explants from 37 (88%) out of 42 differentiated adventitious buds and marker-free transgenic plants containing the GUS gene from five (14%) out of 37 ipt-shooty lines. Furthermore, the GST-MAT vector also induced two marker-free transgenic plants without the production of ipt-shooty intermediates. Southern blot analysis showed that six (86%) out of seven marker-free transgenic plants had a single GUS gene. This result suggests that the GST-MAT vector is useful to generate high frequency, marker-free transgenic plants containing a single transgene.     

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.     

V(D)J recombination and the transgenic brain blues.

The existence of somatic, site-specific recombination in the central nervous system (CNS) has long been hypothesized but has been difficult to investigate experimentally. The finding that RAG-1, which is thought to encode a component of the site-specific recombination machinery of the immune system, is transcribed in the central nervous system (J.J.M. Chun et al., 1991, Cell 64:189-200), has renewed interest in this issue. Two groups (M. Kawaichi et al., 1991, J Biol Chem 266:18,376-18,394; M. Matsuoka et al., 1991, Science 254:81-86) have now reported the results of transgenic mouse experiments designed to determine whether cells of the CNS can perform a site-specific recombination reaction similar to that of lymphocytes. Despite extensive similarities in the design of the two experiments, they yielded discordant results and contradictory conclusions. An analysis of the two studies suggests some explanations for the discrepancies and leads us to two conclusions: first, that the CNS does not carry out the same somatic, site-specific recombination reaction as is found in the immune system and, second, that the question of whether other site-specific recombination processes occur in the brain remains open and largely unaddressed.     

Z/AP, a double reporter for cre-mediated recombination

The Cre/loxP site-specific recombination system combined with embryonic stem cell-mediated technologies has greatly expanded our capability to address normal and disease development in mammals using genetic approaches. The success of this emerging technology hinges on the production of Cre-expressing transgenic lines that provide cell type-, tissue-, or developmental stage-specific recombination between loxP sites placed in the genome. Here we describe and characterize the production of a double-reporter mouse line that provides a convenient and reliable readout of Cre recombinase activity. Throughout all embryonic and adult stages, the transgenic animal expresses the lacZ reporter gene before Cre-mediated excision occurs. Cre excision, however, removes the lacZ gene, allowing expression of the second reporter, the human alkaline phosphatase gene. This double-reporter transgenic line is able to indicate the occurrence of Cre excision in an extremely widespread manner from early embryonic to adult lineages. 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.     

利用热诱导的位点专一性重组系统在烟草中控制基因表达

采用热激启动子Gmhsp17．5C控制Cre定位重组酶介导的DNA删除系统。在这个系统中,在热激启动子控制下的Cre重组酶的表达导致两侧带有相同方向loxp位点的CaMV35S—GUS片段从转基因烟草(Nicotiana tabacum L.cv.W38)的基因组中删除。通过定量PCR的方法鉴定这个转基因系统,显示了这个系统的重组效率。结果显示在两个小时热激处理后转基因烟草中有41％的CaMV35S—Gus片段被删除。由于热激诱导的定点重组系统有容易操作、对热敏感和无背景表达等优点,因此有利于采用这个系统在转基因植物中进行可诱导的基因操作。     

利用热诱导的位点专一性重组系统在烟草中控制基因表达

采用热激启动子Gmhsp17.5C控制Cre定位重组酶介导的DNA删除系统.在这个系统中,在热激启动子控制下的Cre重组酶的表达导致两侧带有相同方向loxp位点的CaMV35S-GUS片段从转基因烟草(Nicotiana tabacum L.cv.W38)的基因组中删除.通过定量PCR的方法鉴定这个转基因系统,显示了这个系统的重组效率.结果显示在两个小时热激处理后转基因烟草中有41%的CaMV35S-GUS片段被删除.由于热激诱导的定点重组系统有容易操作、对热敏感和无背景表达等优点,因此有利于采用这个系统在转基因植物中进行可诱导的基因操作.     

Excision of a selectable marker in transgenic rice (Oryza sativa L.) using a chemically regulated Cre/loxP system

Removal of a selectable marker gene from genetically modified (GM) crops alleviates the risk of its release into the environment and hastens the public acceptance of GM crops. Here we report the production of marker-free transgenic rice by using a chemically regulated, Cre/loxP-mediated site-specific DNA recombination in a single transformation. Among 86 independent transgenic lines, ten were found to be marker-free in the T₀ generation and an additional 17 lines segregated marker-free transgenic plants in the T₁ generation. Molecular and genetic analyses indicated that the DNA recombination and excision in transgenic rice were precise and the marker-free recombinant T-DNA was stable and heritable.The first two authors contributed equally to the work     

Targeted somatic mutagenesis in mouse epidermis

Gene targeting in the mouse is a powerful tool to study mammalian gene function. The possibility to efficiently introduce somatic mutations in a given gene, at a chosen time and/or in a given cell type will further improve such studies, and will facilitate the generation of animal models for human diseases. To create targeted somatic mutations in the epidermis, we established transgenic mice expressing the bacteriophage P1 Cre recombinase or the tamoxifen-dependent Cre-ER(T2) recombinase under the control of the human keratin 14 (K14) promoter. We show that LoxP flanked (floxed) DNA segments were efficiently excised in epidermal keratinocytes of K14-Cre transgenic mice. Furthermore, Tamoxifen administration to adult K14-Cre-ER(T2) mice efficiently induced recombination in the basal keratinocytes, whereas no background recombination was detected in the absence of ligand treatment. These two transgenic lines should be very useful to analyse the functional role of a number of genes expressed in keratinocytes.     

利用FLP/frt重组系统产生无选择标记的转基因烟草植株

在植物转基因植株产生过程中,对转化细胞进行抗性筛选是通用程序,转化细胞的抗性一般是抗生素抗性或除草剂抗性,将赋予转化细胞抗性的选择标记基因删除是提高转基因植物生物安全性的重要措施。来自于啤酒酵母的FLP/frt位点特异性重组系统可有效删除同向定点重组位点frt之间的基因。通过多步骤重组,建立了可在植物中广泛应用的FLP/frt位点特异性重组系统。该系统包括含有frt位点的植物表达载体pCAMBIA1300-betA-frt-als-frt和含有由热诱导启动子hsp启动的FLP重组酶基因的植物表达载体pCAMBIA1300-hsp-FLP-hpt。利用二次转化的方式将二者先后转入烟草植株,热激处理后,热诱导型启动子hsp调控的重组酶FLP基因的表达催化位于选择标记基因als两侧同向frt位点间的重组反应,有效地删除了选择标记基因als。41%的经热激处理的二次转化植株发生了选择标记基因的删除,表明该系统在获得无选择标记基因的转基因植株中有很好的应用价值。     

The chicken HPRT gene: a counter selectable marker for the DT40 cell line

We describe the cloning, characterisation and chromosomal mapping of the chicken hprt gene together with the construction of two counter selectable hprt-/- DT40 derived cell lines. One of these cell lines contains a stably integrated gene encoding a conditionally active cre recombinase and thus allows efficient manipulation of targeted loci by site-specific recombination. These cell lines will enhance the utility of the hyper-recombinogenic DT40 cell line as a system for the genetic analysis of cell autonomous functions in vertebrates and as a tool for mammalian chromosome engineering.