A highly efficient ligand-regulated Cre recombinase mouse line shows that LoxP recombination is position dependent

Conditional gene inactivation using the Cre/loxP system is widely used, but the difficulty in properly regulating Cre expression remains one of the bottlenecks. One approach to regulate Cre activity utilizes a mutant estrogen hormone-binding domain (ER^T) to keep Cre inactive unless the non-steroidal estrogen analog 4-hydroxytamoxifen (OHT) is present. Here we describe a mouse strain expressing Cre-ER^T from the ubiquitously expressed ROSA26 (R26) locus. We demonstrate efficient temporal and spatial regulation of Cre recombination in vivo and in primary cells derived from these mice. We show the existence of marked differences in recombination frequencies between different substrates within the same cell. This has important consequences when concurrent switching of multiple alleles within the same cell is needed, and highlights one of the difficulties that may be encountered when using reporter mice as indicator strains.     

Cre recombinase induces DNA damage and tetraploidy in the absence of LoxP sites

The spatiotemporal manipulations of gene expression by the Cre recombinase (Cre) of bacteriophage P1 has become an essential asset to understanding mammalian genetics. Accumulating evidence suggests that Cre activity can, in addition to excising targeted loxP sites, induce cytotoxic effects, including abnormal cell cycle progression, genomic instability, and apoptosis, which can accelerate cancer progression. It is speculated that these defects are caused by Cre-induced DNA damage at off-target sites. Here we report the formation of tetraploid keratinocytes in the epidermis of keratin 5 and/or keratin 14 promoter-driven Cre (KRT5- and KRT14-Cre) expressing mouse skin. Biochemical analyses and flow cytometry demonstrated that Cre expression also induces DNA damage, genomic instability, and tetraploidy in HCT116 cells, and live-cell imaging revealed an extension of the G₂ cell cycle phase followed by defective or skipping of mitosis as cause for the tetraploidy. Since tetraploidy eventually leads to aneuploidy, a hallmark of cancer, our findings highlight the importance of distinguishing non-specific cytopathic effects from specific Cre/loxP-driven genetic manipulations when using Cre-mediated gene deletions.     

Expression of Cre recombinase during transient phage infection permits efficient marker removal in Streptomyces

We report a system for the efficient removal of a marker flanked by two loxP sites in Streptomyces coelicolor, using a derivative of the temperate phage C31 that expresses Cre recombinase during a transient infection. As the test case for this recombinant phage (called Cre-phage), we present the construction of an in-frame deletion of a gene, pglW, required for phage growth limitation or Pgl in S.coelicolor. Cre-phage was also used for marker deletion in other strains of S.coelicolor.     

Construction of a long hairpin RNA expression library using Cre recombinase

A large number of genome-wide screens using RNA interference (RNAi) libraries have been utilized to determine the function of individual gene products involved in a variety of biological processes. In this study, we describe a new method to enzymatically generate a long hairpin RNA (lhRNA) expression library from a cDNA plasmid library using a nicking endonuclease, BcaBEST DNA polymerase, and Cre recombinase without excising the inserted DNA fragment from the plasmid vector. This method involves 5 steps: (1) conversion of an inserted DNA fragment in a plasmid into a direct repeat (DR); (2) purification of the plasmid containing the DR; (3) subcloning a lox71 cassette into the plasmid; (4) conversion of the DR in the plasmid into an inverted repeat (IR) using Cre recombinase; and (5) purification of the plasmid containing the IR. We also established an efficient method for inserting DNase I-digested DNA fragments into expression plasmids to enable construction of a cDNA plasmid library suitable as source materials to construct the lhRNA expression library. We confirmed that each of the lhRNA expression plasmids constructed using this method induced strong RNAi in a silkworm cell line, NIAS-Bm-oyanagi2.     

Conditional gene modification in mouse liver using hydrodynamic delivery of plasmid DNA encoding Cre recombinase

The success of Cre-mediated conditional gene targeting in liver of mice has until now depended on the generation of Cre recombinase transgenic mice or on viral-mediated transduction. Here, we sought to establish the feasibility of using hydrodynamic gene delivery of Cre recombinase into liver, using a ROSA26 EGFP mouse. The expression of EGFP and beta-galactosidase was exclusively detected in the liver of mice treated with hydrodynamic gene delivery of Cre recombinase, as assessed with fluorescence microscopy and X-Gal staining, respectively; Southern blotting also showed that Cre mediated recombination occurred specifically in the liver and not in other organs. The Cre mediated recombination reached about 61% of hepatocytes of mouse after repeated injection, as analyzed by flow cytometry. These results demonstrate that Cre recombinase can be transferred to the liver of mice through a simple hydrodynamic gene-delivery approach and can mediate efficient recombination in hepatocytes. Thus, hydrodynamic gene delivery of the Cre recombinase provides a valuable approach for Cre-loxP-mediated conditional gene modification in the liver of mice.     

Expression of Cre recombinase in pigment cells

Conditional gene targeting using the Cre/loxP system enables specific deletion of a gene in a tissue of interest. For application of Cre-mediated recombination in pigment cells, Cre expression has to be targeted to pigment cells in transgenic mice. So far, no pigment cell-specific Cre transgenic line has been reported and we present and discuss our first results on use of Cre recombinase in pigment cells. A construct was generated where Cre recombinase is controlled by the promoter of the mouse dopachrome tautomerase (Dct) gene. The construct was functionally tested in vitro and introduced into mice. Following breeding to two reporter mouse strains, we detected Cre recombinase activity in telencephalon, melanoblasts, and retinal pigment epithelium (RPE). Our data demonstrate the feasibility of pigment cell-specific Cre/loxP-mediated recombination.     

Establishment of Cre/LoxP recombination system in transgenic rats

The rat has offered an important animal model in biomedical research including surgical procedure. However, advanced genetic manipulation has progressed less far in the rat than in the mouse. Here we report the Cre/LoxP transgenic rat system, demonstrating conditional chromosomal translocation both in the fertilization and adult stage, spatio-temporal gene controlling by catheter-based adenoviral gene transfer, and muscular fusion events in the limb transplant. Taking advantage of the larger body size of the rat than the mouse, this rat system provides a potential value to evaluate biomedical and therapeutic significance for gene therapy and regenerative medicine.     

Inducible expression of V600EBraf using tyrosinase-driven Cre recombinase results in embryonic lethality

We recently demonstrated that expression of ^V600EBraf in mature mouse melanocytes induces melanoma. Here, we show that expression of ^V600EBraf using the tyrosinase promoter leads to an unexpected embryonic lethality, with the animals dying before, at, or shortly after birth. The mice suffer from a range of developmental defects in the skin, the brain, the eyes and the heart, tissues that are normally colonized by melanocytes. We show that the ^V600EBraf expressing cells are potential melanocytic precursors that are fully transformed, suggesting that ^V600EBraf stimulates proliferation and blocks differentiation of these cells. Our data suggests that the presence of these cells in the organs that are normally occupied by melanocytes leads to severe developmental disruption, resulting in catastrophic defects and leading to death of the individual.     

Functional expression of the Cre recombinase in actinomycetes

Site-specific recombinases revolutionized “in vivo” genetic engineering because they can catalyze precise excisions, integrations, inversions, or translocations of DNA between their distinct recognition target sites. We have constructed a synthetic gene encoding Cre recombinase with the GC content 67.7% optimized for expression in high-GC bacteria and demonstrated this gene to be functional in Streptomyces lividans. Using the synthetic cre(a) gene, we have removed an apramycin resistance gene flanked by loxP sites from the chromosome of S. lividans with 100% efficiency. Sequencing of the chromosomal DNA part showed that excision of the apramycin cassette by Cre recombinase was specific.     

利用Cre/LoxP系统删除转基因山羊体内的选择标记基因

在制备转基因家畜过程中的一个关键步骤是使用选择标记基因 (Selectable marker genes,SMGs) 将转基因整合细胞从大量的正常细胞中筛选出来,这导致了SMGs整合入家畜的基因组内持续传递给后代。SMGs已被证明能够显著影响基因组内整合位点处的基因调控,也增加了对转基因动物安全评价的复杂性。为了确定转基因山羊制备过程中SMGs的删除时机和删除方法,在体细胞克隆前后两个时段内,利用Cre/loxP系统删除SMGs的可行性,同时比较了蛋白转导和质粒共转染两种Cre导入方式的删除效率。结果表明：尽管在首次对山羊成纤维细胞进行遗传修饰后即可进行SMGs删除,但两次遗传修饰导致细胞严重老化,无法用于后续的体细胞克隆羊制备。在转基因山羊的成体细胞中删除SMGs不存在上述问题,成功率高,缺点是试验周期长、耗资增大。Cre表达质粒瞬时转染能够删除SMGs,但有超过30%的无SMGs细胞克隆中整合有质粒序列。TAT-CRE蛋白质转导方法可以避免引入的新外源基因,SMGs删除率达到43.9%~72.8%,是一种较佳的SMGs删除手段。     

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.     

Cre/LoxP系统在转基因小鼠上的应用策略

Cre/LoxP位点特异重组酶系统已发展为在体内外进行遗传操作的一个新的有力工具.该系统在转基因小鼠上的应用,可使转基因的表达或靶基因的缺失/突变的位点特异DNA重组不仅发生在小鼠发育的某一阶段或特定的组织器官,而且,若与控制Cre表达或功能的诱导系统结合,则可以时空方式体现.这些基于重组的策略可能对基因功能的研究和人类疾病的动物模型的建立产生深刻影响.     

Cre/LoxP重组系统的改造及在树干毕赤酵母中的应用

为了实现在P.stipitis中进行无痕基因敲除,以Cre/LoxP系统为研究对象,首先通过同源重组构建尿嘧啶营养缺陷型树干毕赤酵母(ura3-);同时通过定点突变pSH47-Hpt质粒的hpt基因和cre基因,将CDS区CTG突变为TTG;最后以乙醛脱氢酶基因为靶基因,验证突变后的Cre/LoxP系统在P.stipitis进行无痕基因敲除的可行性。结果表明:本文在P.stipitis中成功使用潮霉素B抗性标记,经过修饰后的Cre/LoxP敲除系统能够在P.stipitis中无痕敲除目的基因,为后续研究P.stipitis功能基因和改造代谢途径提供了一种试验方法和筛选标记。     

Highly effective removal of floxed Blasticidin S resistance cassettes from Dictyostelium discoideum mutants by extrachromosomal expression of Cre

The inactivation of proteins in cells is inevitable to study their physiological role in various cellular processes. In contrast to strategies to alter the amount of active proteins in cells, only a gene knockout guarantees complete removal of the protein of interest. For Dictyostelium discoideum cells, the gene replacement construct typically consists of a Blasticidin S resistance (Bsr) cassette flanked by fragments of the target gene to allow insertion by homologous recombination. More advanced knockout constructs additionally carry loxP sites on both sides of the Bsr cassettes for subsequent removal of the selection marker by transient expression of Cre recombinase, thus allowing generation of multiple knockouts using just a single selection marker. However, due to its design, the available neomycin selection-based Cre expression plasmid occasionally tends to integrate into the genome and also yield only a moderate number of transfectants in liquid media. In some cases, for instance in SCAR-null cells, it was not possible to remove the Bsr cassette without stable integration of the Cre expression vector into the genome. To circumvent these difficulties we designed the extrachromosomal Cre-recombinase expression vector pTX-NLS-Cre. We verified the greatly improved efficacy of this novel Cre-loxP approach by removal of the Bsr cassette in five different cell lines including the SCAR-null mutant. As a consequence, this vector will be a highly valuable means for the rapid generation of single or multiple mutants remaining sensitive to the most reliable selection markers Blasticidin S and neomycin.     

Efficient removal of loxP-flanked DNA sequences in a gene-targeted locus by transient expression of Cre recombinase in fertilized eggs

The bacteriophage P1 Cre/loxP site-specific recombination system is a useful tool for engineering chromosomal changes in animal cells. Transient expression of the Cre recombinase gene directly introduced into fertilized eggs by pronuclear injection has been reported to provide an efficient method of transgene modulation in fertilized eggs. In the present study, we examined the efficacy of this method to remove loxP-flanked DNA sequences in a gene-targeted locus in fertilized eggs. We replaced a part of the T-cell receptor γ (TCR Vγ) locus with homologous sequences containing a loxP-flanked neogene in mouse embryonic stem (ES) cells by gene-targeting technique. The resulting ES cell clones containing the mutant allele (Vγ^LNL) were used to generate chimeric mice by blastocyst injection. Eight male chimeras were bred with superovulated wild-type female mice. One hundred and seventy-six fertilized eggs were collected, and subjected to pronuclear injection of the Cre expression plasmid, pCAGGS-Cre, of a covalently closed circular form. Three out of 11 pups inherited the targeted Vγ locus. The inherited targeted allele of these 3 mice was shown to have undergone Cre-mediated recombination, resulting in a deletion of the loxP-flanked sequences (Vγ^Δ) as shown by Southern blot analysis of DNA from tail biopsies. All 3 founder mutant mice were capable of transmitting the Vγ^Δ locus to their offspring. The other 8 pups carried only wild-type alleles. There were no pups carrying the unrecombined Vγ^LNL locus. Thus, the frequency of Cre-mediated recombination was 100% (3/3) with this method. In contrast, when closed circular pCAGGS-Cre plasmid was introduced into ES cells by electroporation, the recombination frequency of the Vγ^LNL locus was 9.6%. These results indicated that our system based on transient expression of the Cre recombinase gene directly introduced into fertilized eggs by pronuclear injection provides a fast and efficient method for generating mutant mice with desired deletions or translocations in target genes. Mol Reprod Dev 46:109–113, 1997. © 1997 Wiley-Liss, Inc.     

Temporal regulation of Cre recombinase activity in neural stem cells

Neural stem cells are known to give rise to distinct subtypes of neurons and glial cells over time by changing their competency. However, precise characterization of neural stem cells at various developmental stages remains to be performed. For such analysis, a tool to manipulate neural stem cells at different time points is necessary. Here, we generated transgenic mice that express Cre-ER(T2) in the ventricular zone of the developing nervous system under the control of the nestin promoter and enhancer (Nes-CreER(T2)). In mice expressing Cre-ER(T2) at appropriate levels, Cre recombinase activity was mostly inactive but efficiently activated by tamoxifen within 1 day. When such mice were crossed with the ROSA-26 or Z/EG reporter mice, neural stem cells were permanently labeled after administration of tamoxifen. Thus, Nes-CreER(T2) mice offer a powerful tool to manipulate neural stem cells genetically at desired time points.     

Highly restricted expression of Cre recombinase in cerebellar Purkinje cells

The Purkinje neuron, one of the most fascinating components of the cerebellar cortex, is involved in motor learning, motor coordination, and cognitive function. Purkinje cell protein 2 (Pcp2/L7) expression is highly restricted to Purkinje and retinal bipolar cells, where it has been exploited to enable highly specific, Cre recombinase-mediated, site-specific recombination. Previous studies showed that mice carrying a Cre transgene produced by insertion of Cre cDNA into a small 2.88-kb Pcp2 DNA fragment expressed Cre in Purkinje cells; however, some Cre activity was also observed outside the target tissues. Here, we used Red-mediated recombineering to insert Cre cDNA into a 173-kb BAC carrying the entire intact Pcp2 gene, and characterize the resultant BAC/Cre transgenic mice for Cre expression. We show that BAC/Cre transgenic mice have exclusive Cre expression in Purkinje and bipolar cells and nowhere else. These mice will facilitate Purkinje cell and retinal bipolar cell-specific genetic manipulation.