Meox1Cre: a mouse line expressing Cre recombinase in somitic mesoderm

We developed a novel strategy based on in vitro DNA transposition of phage Mu to construct vectors for "knock-in" of the gene encoding Cre recombinase into endogenous loci in embryonic stem cells. This strategy was used to introduce Cre into the mouse Meox1 locus, which was expected to drive Cre expression in the presomitic and somitic mesoderm. In embryos heterozygous for both Meox1(Cre) and R26R or Z/AP reporter alleles, specific and efficient recombination of the reporter alleles was detected in the maturing somites and their derivatives, including developing vertebrae, skeletal muscle, back dermis, as well as endothelium of the blood vessels invading the spinal cord and developing limbs. In contrast to the somitic mesoderm, Cre activity was not observed in the cranial paraxial mesoderm. Thus, the Meox1(Cre) allele allows detailed fate-mapping of Meox1-expressing tissues, including derivatives of the somitic mesoderm. We used it to demonstrate dynamic changes in the composition of the mesenchyme surrounding the developing inner ear. Meox1(Cre) may also be used for tissue-specific mutagenesis in the somitic mesoderm and its derivatives.     

他莫昔芬诱导的Cre重组酶在hGfapCreERT2转基因鼠小脑中的表达研究

目的探讨他莫昔芬诱导的hGfapCreERT2转基因鼠小脑中表达Cre重组酶的细胞类型。方法 hGfapCre-ERT2/Rosa26R转基因小鼠在胚胎晚期和出生早期用他莫昔芬诱导Cre重组酶表达,对小脑组织切片行X-gal染色,然后用细胞种类特异性抗体进行免疫组织化学染色,并和X-gal染色双重标记。结果在出生后第7天(P7)、第14天(P14)和第60天(P60),X-gal阳性染色和胶质细胞抗体Blbp阳性染色共标记,和神经元抗体Neun、浦肯野细胞抗体Calbindin及少突胶质细胞前体细胞抗体NG2不共标。结论自胚胎晚期第17.5天(E17.5)后用他莫昔芬诱导hGfapCreERT2转基因鼠,发现Cre重组酶特异性在小脑星形胶质细胞中表达,不在神经元、浦肯野细胞、少突胶质细胞前体细胞中表达。     

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.     

Expression of Cre recombinase in early diploid trophoblast cells of the mouse placenta

An increasing number of genes known to be critical for cell cycle control, differentiation, and tumor suppression have been found to impact development of the placenta. To elucidate how these genes contribute to development of embryonic and extra-embryonic lineages, we generated a transgenic mouse in which the Cre transgene is driven by placenta-specific regulatory sequences from the human CYP19 gene. Using ROSA26 conditional reporter mice, we could detect expression of the CYP19-Cre transgene throughout the extra-embryonic ectoderm and in the ectoplacental cone at embryonic day 6.5 (E6.5). By E11.5, recombination of LoxP reporter sites was detected in all derivatives of trophoblast stem cells, including spongiotrophoblast, giant cells, and labyrinth trophoblasts. We conclude that the CYP19-Cre transgenic mouse developed here can be used in combination with conditional alleles to distinguish between embryonic and extra-embryonic gene function, and to begin to map the period of time when gene function is critical during development.     

Generation of a mouse line expressing Cre recombinase in glycinergic interneurons

In caudal regions of the CNS, glycine constitutes the major inhibitory neurotransmitter. Here, we describe a mouse line that expresses Cre recombinase under the control of a BAC transgenic glycine transporter 2 (GlyT2) promoter fragment. Mating of GlyT2‐Cre mice with the Cre reporter mouse lines Rosa26/LacZ and Rosa26/YFP and analysis of double transgenic offsprings revealed strong transgene activity in caudal regions of the central nervous system, i.e., brain stem and spinal cord. Some additional Cre expression was observed in cortical and cerebellar regions. In brain stem and spinal cord, Cre expressing cells were identified as glycinergic interneurons by staining with GlyT2‐ and glycine‐immunoreactive antibodies; here, >80% of the glycine‐immunoreactive cells expressed the Cre reporter protein. These data indicate that GlyT2‐Cre mice are a useful tool for the genetic manipulation of glycinergic interneurons. genesis 48:437–445, 2010. © 2010 Wiley‐Liss, Inc.     

Pan-myocardial expression of Cre recombinase throughout mouse development

Mouse-lines expressing Cre recombinase in a tissue-specific manner are a powerful tool in developmental biology. Here, we report that a 3 kb fragment of the Xenopus laevis myosin light-chain 2 (XMLC2) promoter drives Cre recombinase expression in a cardiac-restricted fashion in the mouse embryo. We have isolated two XMLC2-Cre lines that express recombinase exclusively within cardiomyocytes, from the onset of their differentiation in the cardiac crescent of the early embryo. Expression is maintained throughout the myocardium of the embryonic heart tube and subsequently the mature myocardium of the chambered heart. Recombinase activity is detected in all myocardial tissue, including the pulmonary veins. One XMLC2-Cre line shows uniform expression while the other only expresses recombinase in a mosaic fashion encompassing less than 50% of the myocardial cells. Both lines cause severe cardiac malformations when crossed to a conditional Tbx5 line, resulting in embryonic death at midgestation. Optical projection tomography reveals that the spectrum of developmental abnormalities includes a shortening of the outflow tract and its abnormal alignment, along with a dramatic reduction in trabeculation of the ventricular segment of the looping heart tube.     

Codon-improved Cre recombinase (iCre) expression in the mouse

By applying the mammalian codon usage to Cre recombinase, we improved Cre expression, as determined by immunoblot and functional analysis, in three different mammalian cell lines. The improved Cre (iCre) gene was also designed to reduce the high CpG content of the prokaryotic coding sequence, thereby reducing the chances of epigenetic silencing in mammals. Transgenic iCre expressing mice were obtained with good frequency, and in these mice loxP-mediated DNA recombination was observed in all cells expressing iCre. Moreover, iCre fused to two estrogen receptor hormone binding domains for temporal control of Cre activity could also be expressed in transgenic mice. However, Cre induction after administration of tamoxifen yielded only low Cre activity. Thus, whereas efficient activation of Cre fusion proteins in the brain needs further improvements, our studies indicate that iCre should facilitate genetic experiments in the mouse.     

Cre重组酶表达载体的构建及其在转基因小鼠胰腺中的表达

组织特异性表达Cre重组酶的转基因小鼠是进行组织特异性条件敲除研究的关键。采用PCR扩增大鼠胰岛素基因705bp启动子指导发胰岛细胞中特异表达;同时采用改构的Cre重组酶基因,在其5'端添加有真核核糖体结合序列和核定位序列使Cre重组酶能穿越核膜在细胞核能发挥功能;同时,为了保证原核基因Cre能在真核系统顺利表达,在其3'端添加含内含子的人生长激素基因。构建的表达载体在去除原核序列后用显微注射方法转基因小鼠,在出生的27只仔鼠中,PCR检测共获得7只Cre整合阳性的转基因小鼠,整合率26％。这种Cre转基因小鼠与基因组小携带LoxP位点的条件基因打靶小鼠交配,在胰腺组织中可以检测到Cre介导的重组,表明Cre在转基因小鼠胰腺中有表达。     

Functional expression of Cre recombinase in sub-regions of mouse CNS and retina

We describe a strategy for generating CNS and retina sub-region-specific mutations using the Cre/loxP system. Transgenic mice expressing Cre recombinase under the control of the c-kit promoter were established. Functional Cre expression was predominantly found to be restricted to the CA1, CA2 and CA3 regions of the hippocampus, the anterior region of the dentate gyrus, and to the ganglion cell layer of the retina.     

Generation of a transgenic mouse model with chondrocyte-specific and tamoxifen-inducible expression of Cre recombinase

Postnatal cartilage development and growth are regulated by key growth factors and signaling molecules. To fully understand the function of these regulators, an inducible and chondrocyte-specific gene deletion system needs to be established to circumvent the perinatal lethality. In this report, we have generated a transgenic mouse model (Col2a1-CreER(T2)) in which expression of the Cre recombinase is driven by the chondrocyte-specific col2a1 promoter in a tamoxifen-inducible manner. To determine the specificity and efficiency of the Cre recombination, we have bred Col2a1-CreER(T2) mice with Rosa26R reporter mice. The X-Gal staining showed that the Cre recombination is specifically achieved in cartilage tissues with tamoxifen-induction. In vitro experiments of chondrocyte cell culture also demonstrate the 4-hydroxy tamoxifen-induced Cre recombination. These results demonstrate that Col2a1-CreER(T2) transgenic mice can be used as a valuable tool for an inducible and chondrocyte-specific gene deletion approach.     

Rapid conversion of EUCOMM/KOMP-CSD alleles in mouse embryos using a cell-permeable Cre recombinase

We describe here use of a cell-permeable Cre to efficiently convert the EUCOMM/KOMP-CSD tm1a allele to the tm1b form in preimplantation mouse embryos in a high-throughput manner, consistent with the requirements of the International Mouse Phenotyping Consortium-affiliated NIH KOMP2 project. This method results in rapid allele conversion and minimizes the use of experimental animals when compared to conventional Cre transgenic mouse breeding, resulting in a significant reduction in costs and time with increased welfare benefits.     

Expression and activity of osteoblast-targeted Cre recombinase transgenes in murine skeletal tissues

The Cre/loxP recombination system can be used to circumvent many of the limitations of generalized gene ablation in mice. Here we present the development and characterization of transgenic mice in which Cre recombinase has been targeted to cells of the osteoblast lineage with 2.3 kb (Col 2.3-Cre) and 3.6 kb (Col 3.6-Cre) fragments of the rat Col1a1 promoter. Cre mRNA was detected in calvaria and long bone of adult Col 2.3-Cre and Col 3.6-Cre mice, as well as in tendon and skin of Col 3.6-Cre mice. To obtain a historical marking of the temporal and spatial pattern of Cre-mediated gene rearrangement, Col-Cre mice were bred with ROSA26 (R26R) mice in which Cre-mediated excision of a floxed cassette results in LacZ expression. In Col 2.3-Cre;R26R and Col 3.6-Cre;R26R progeny, calvarial and long bone osteoblasts showed intense beta-gal staining at embryonic day 18 and postnatal day 5. The spatial pattern of beta-gal staining was more restricted in bone and in bone marrow stromal cultures established from Col 2.3-Cre;R26R mice. Similar differences in the spatial patterns of expression were seen in transgenic bone carrying Col1a1-GFP visual reporters. Our data suggest that Col 2.3-Cre and Col 3.6-Cre transgenic mice may be useful for conditional gene targeting in vivo or for obtaining osteoblast populations for in vitro culture in which a gene of interest has been inactivated.     

Transgenic mouse lines expressing Cre recombinase specifically in posterior notochord and notochord

During development, the organizer provides instructive signals to surrounding cells as well as contributing cells to axial structures. To dissect organizer function at different developmental stages, conditional approaches such as the Cre/loxP system for conditional mutagenesis are particularly useful. Here we describe two new Cre transgenic mouse lines, Foxa2 NFP-Cre and Nodal PNC-Cre, with activity in two organizer domains, the posterior notochord (PNC) and notochord. These lines were made using defined regulatory elements from the Foxa2 and Nodal genes that direct Cre expression in overlapping domains of the PNC and notochord. Our detailed analysis of the timing and location of Foxa2 NFP-Cre and Nodal PNC-Cre activity indicates that these lines are appropriate for conditional mutagenesis of genes expressed from early somite stages onward.     

Expression of Cre recombinase in chondrocytes causes abnormal craniofacial and skeletal development

The cranial base synchondroses are growth centers that drive cranial and upper facial growth. The intersphenoid synchondrosis (ISS) and the spheno-occipital synchondrosis (SOS) are two major synchondroses located in the middle of the cranial base and are maintained at early developmental stages to sustain cranial base elongation. In this study, we report unexpected premature ossification of ISS and SOS when Cre recombinase is activated in a chondrocyte-specific manner. We used a Cre transgenic line expressing Aggrecan enhancer-driven, Tetracycline-inducible Cre (ATC), of which expression is controlled by a Col2a1 promoter. Neonatal doxycycline injection or doxycycline diet fed to breeders was used to activate Cre recombinase. The premature ossification of ISS and/or SOS led to a reduction in cranial base length and subsequently a dome-shaped skull. Furthermore, the mice carrying either heterozygous or homozygous conditional deletion of Tsc1 or Fip200 using ATC mice developed similar craniofacial abnormalities, indicating that Cre activity itself but not conditional deletion of Tsc1 or Fip200 gene, is the major contributor of this phenotype. In contrast, the Col2a1-Cre mice carrying Cre expression in both perichondrium and chondrocytes and the mice carrying the conditional deletion of Tsc1 or Fip200 using Col2a1-Cre did not manifest the same skull abnormalities. In addition to the defective craniofacial bone development, our data also showed that the Cre activation in chondrocytes significantly compromised bone acquisition in femur. Our data calls for the consideration of the potential in vivo adverse effects caused by Cre expression in chondrocytes and reinforcement of the importance of including Cre-containing controls to facilitate accurate phenotype interpretation in transgenic research.

     

Inducible Cre recombinase activity in mouse mature astrocytes and adult neural precursor cells

Two transgenic mouse lines expressing an inducible form of the Cre recombinase (CreER(TM)) under the control of the human GFAP promoter have been generated and characterized. In adult mice, expression of the fusion protein is largely confined to astrocytes in all regions of the central nervous system. Minimal spontaneous Cre activity was detected and recombination was efficiently induced by intraperitoneal administration of tamoxifen in adult mice. The pattern of recombination closely mirrored that of transgene expression. The percentage of astrocytes undergoing recombination varied from region to region ranging from 35% to 70% while a much smaller portion (<1%) of oligodendrocytes and neural precursor cells showed evidence of Cre activity. These mouse lines will provide important tools to dissect gene function in glial cells and in gliomagenesis.     

Cre stoplight: a red/green fluorescent reporter of Cre recombinase expression in living cells.

The Cre/lox system is a powerful genetic tool with which to manipulate the genome. Here, we describe the development of a simple reporter system for Cre recombinase, called the Cre Stoplight. In the absence of Cre, the red fluorescent protein is expressed; when Cre catalyzes a recombination event, the green fluorescent protein is produced. Testing this system in transiently transfected cells showed that it produced robust signals (90% of the cells converted from red to green) when equal amounts of the plasmids encoding Cre recombinase and the Cre Stoplight were used. A 1:100 ratio of enzyme to reporter plasmid produced similar results, and a 1:10000 ratio was necessary to significantly reduce the number of cells converting to green (1%).     

Lack of effect of maternal age on UV-induced DNA repair in mouse oocytes

Oocytes at the dictyate stage young (8–14 weeks) and old (12–15 months) BALB/c mice were manually isolated and UV-irrdiated. They were cultured for 1 h in medium containing tritiated thymidine and chased for a furthur hour in cold thymidine medium before being incubated for 18–20 h in medium with no added thymidine. Oocytes which had developed to metaphase II were analysed following autoradiography. Pooled results from 14 replicate experiments revealed no significant age-related difference between the mean corrected grain count per cell [159.2 ± 8.5 (86 cells) for young mice and 164.6 ± 9.8 (70 cells) for the old animals]. Thus in the female mouse the oocyte's capacity to repair UV-induced damage is apparently maintained at a high level throughout reprodcutive life.