Brief expression of a GFP cre fusion gene in embryonic stem cells allows rapid retrieval of site-specific genomic deletions.

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.     

Primary Spermatocyte-Specific Cre Recombinase Activity in Transgenic Mice

We have evaluated the specificity of Cre recombinase activity in transgenic mice expressing Cre under the control of the synatonemal complex protein 1 (Sycp1) gene promoter. Sycp1Cre mice were crossed with the ROSA26 reporter line R26R, to monitor the male germ cell stage-specificity of Cre activity as well as to verify that Cre was not active previously during development of other tissues. X-gal staining detected Cre-mediated recombination only in testis. Detailed histological examination indicated that weak Cre-mediated recombination occurred as early as in zygotene spermatocytes at stage XI of the cycle of the seminiferous epithelium. Robust expression of X-gal was detected in early to mid-late spermatocytes at stages V-VIII. We conclude that this transgenic line is a powerful tool for deleting genes of interest specifically during male meiosis.     

Cre/lox介导剔除转双价抗虫sck+cryIAc基因籼稻恢复系明恢86材料的选择标记基因

Cre/lox系统通过其Cre重组酶对lox序列进行切割和重新连接,介导lox序列发生特异性重组.利用重组报告基因系统Pactin-lox-hpt-lox-gusA,对Cre/lox系统在水稻(Oryzasativa L.)中介导转基因的剔除进行了研究.Pactin-lox-hpt-lox-gusA系统中选择标记hpt基因侧翼含两个同向lox位点,并位于水稻actinl启动子和gusA基因之间.当hpt在Cre酶作用下被剔除时,actinl启动子可以和gusA基因融合在一起从而驱动GUS表达.通过农杆菌介导获得了分别转cre基因、Pactin-lox-hpt-lox-gusA结构和双价抗虫基因lox-hpt-lox-sck-cryIAc结构的水稻.利用有性杂交方法将cre基因导入到转化lox结构的植株中.在4个转Pactin-lox-hpt-lox-gusA T0植株×转cre T0植株所配组合的30个杂交F1植株中,12个植株表达GUS活性,9个表现潮霉素敏感,表明hpt基因被剔除.研究进一步通过Cre/lox介导剔除转双价抗虫sck cryIAc基因籼稻恢复系明恢86材料基因组中的选择标记hpt基因.在9个转lox-hpt-lox-sck-cryIAcT2代纯合植株×转creT2代纯合植株所配组合的77个杂交F1植株中,56个植株表现潮霉素敏感.分子分析证实在这些对潮霉素敏感的植株中hpt基因已经被剔除.     

利用Cre/loxP系统删除转Bt基因水稻中的选择标记基因

来源于噬菌体P1的Cre/loxP位点特异性重组系统是目前在植物遗传转化中应用较多,较成熟的一个标记基因删除系统。在这个系统中,Cre酶可以特异性的识别和切割位于两个lox位点之间的标记基因,整个系统重组仅需Cre和lox识别位点即可完成而无需其它辅因子的参加。利用农杆菌介导法成功地将cre基因导入供试材料"皖粳97",得到转hpt-cre基因水稻植株;将其与先期转基因育成的携带loxp-hpt-loxp-bt基因的"皖粳97"株系进行田间杂交,通过PCR分析,Cre/loxP重组系统定向删除了潮霉素抗性筛选标记基因。     

Novel recombination system using Cre recombinase alpha complementation

A major limitation for the use of Cre recombinase is its toxicity and a lack of temporal control over its activity. We have developed a new recombination system using Cre recombinase α-complementation. Cre recombinase was divided and one fragment (β) was introduced into cells between two loxP sites with a CMV promoter in the upstream. The gene of interest (EGFP) was positioned just downstream of this construct. Cre recombinase activity was recovered by adding the other part of the molecule (α) to cells as a protein fragment, as evidenced by the expression of EGFP under the control of the CMV promoter. The activity of fragmented cre reached 68% of that of the wild type enzyme at 1 μM α-protein.     

Efficient bicistronic expression of cre in mammalian cells

Cre recombinase-mediated DNA recombination is proving to be a powerful technique for the generation of mosaic mutant mice. To develop this technology further, we have altered the cre gene to enhance its expression in mammalian cells and have tested its efficiency of expression in a bicistronic message. Using a transient transfection assay, we found that the extension of a eukaryotic translation initiation consensus sequence, the insertion of two N-terminal amino acids, and the mutation of a cryptic splice acceptor site did not detectably alter Cre recombinase activity. The addition of either of two introns resulted in an approximately 2-fold increase in recombination frequency. We then tested the relative efficacy of Cre-mediated recombination in several bicistronic messages having the encephalomyocarditis virus internal ribosome entry site (IRES). Recombination frequencies were only reduced 2-fold relative to a comparable monocistronic cre gene. The latter results indicate that it will be possible to generate transgenic mouse strains having tissue-specific expression of the Cre recombinase through integration of an IRES-cre gene without disabling the targeted gene.     

Conditional disruption of ubiquitous calpains in the mouse

Ubiquitous mu- and m-calpain proteases are implicated in development and apoptosis. They are heterodimers consisting of 80-kDa catalytic subunits encoded by capn1 and capn2, respectively, and a common 28-kDa regulatory subunit encoded by capn4. The regulatory subunit is required to maintain stability and activity of mu- and m-calpains; thus, genetic disruption of capn4 was predicted to eliminate both calpain activities. Germline disruption of capn4 caused embryonic lethality, hampering the use of those mouse models to explore physiological calpain functions. Here we describe a loxP/cre conditional capn4 targeted mouse model that enables tissue-specific and temporal deletion of calpain activity. Disruption of the floxed capn4 gene using a ubiquitous cytomegalovirus promoter driven Cre recombinase transgene led to midgestation embryonic lethality. Fibroblasts from these embryos lacked detectable regulatory subunit expression, had reduced levels of the mu- and m-calpain catalytic subunits, and had no detectable mu- and m-calpain activities. These defects were corrected with a capn4-encoding lentivirus.     

A transgenic Cre mouse line for the study of cortical and hippocampal development

Wnt signaling regulates cortical and hippocampal development. In a previous study we found that a particular Wnt receptor, Frizzled9 (Fzd9), was selectively expressed in both the developing and adult hippocampus. Taking advantage of the specificity of this promoter, we generated a transgenic cre mouse line using the putative control elements of the Fzd9 gene. In the Fzd9‐cre mice, Cre is mainly detected in the developing cortex and hippocampus and is confined to the CA fields and dentate gyrus in adults. Furthermore, by crossing the Fzd9‐cre mouse with the ROSA26 reporter line, we examined the activity of Cre and found that it has very high recombination efficiency. Thus, this mouse line will likely prove to be a useful tool for studying cortical and hippocampal development via activation or inactivation of interesting genes. genesis 48:343–350, 2010. © 2010 Wiley‐Liss, Inc.     

利用不相容质粒共转化大肠杆菌对Cre重组酶体内重组活性的可视检测

源自噬菌体P1的Cre重组酶可以识别 34bp的靶DNA序列loxP ,进行位点特异性的重组反应。为了简便地检测Cre酶在大肠杆菌中的重组活性 ,分别将cre基因和上下游带有loxP的绿色荧光蛋白基因 (gfp)克隆到具有不同抗性的两种不相容质粒中 ,然后将构建的原核表达载体pET30a Cre和pET2 3b loxGFP电击共转化大肠杆菌BL2 1(DE3) ,利用卡那霉素和氨苄青霉素双抗生素抗性进行筛选。通过直接观察转化子的绿色荧光 ,便可以显示Cre酶的体内重组活性 ,并进一步通过SDS PAGE分析、质粒酶切鉴定进行了验证。结果表明 :以gfp为报告基因、通过两种不相容质粒共转化大肠杆菌可以为研究和改进Cre loxP重组系统提供一种简便直观的检测方法     

Cre／lox介导剔除转双价抗虫sck＋cryIAc基因籼稻恢复系明恢86材料的选择标记基因

Cre/lox系统通过其Cre重组酶对lox序列进行切割和重新连接,介导lox序列发生特异性重组。利用重组报告基因系统Pactin-lox-hpt-lox-gusA,对Cre/lox系统在水稻(Oryza　sativa　L.)中介导转基因的剔除进行了研究。Pactin-lox-hpt-lox-gusA系统中选择标记hpt基因侧翼含两个同向lox位点,并位于水稻actin1启动子和gusA基因之间。当hpt在Cre酶作用下被剔除时,actin1启动子可以和gusA基因融合在一起从而驱动GUS表达。通过农杆菌介导获得了分别转cre基因、Pactin-lox-hpt-lox-gusA结构和双价抗虫基因lox-hpt-lox-sck-cryIAc结构的水稻。利用有性杂交方法将cre基因导入到转化lox结构的植株中。在4个转Pactin-lox-hpt-lox-gusA　T0植株×转cre　T0植株所配组合的30个杂交F1植株中,12个植株表达GUS活性,9个表现潮霉素敏感,表明hpt基因被剔除。研究进一步通过Cre/lox介导剔除转双价抗虫sck　 cryIAc基因籼稻恢复系明恢86材料基因组中的选择标记hpt基因。在9个转lox-hpt-lox-sck-cryIAc　T2代纯合植株×转creT2代纯合植株所配组合的77个杂交F1植株中,　56个植株表现潮霉素敏感。分子分析证实在这些对潮霉素敏感的植株中hpt基因已经被剔除。     

cre基因在大肠杆菌中的表达及表达蛋白活性的检测

Cre重组酶来自噬菌体P1,可以识别特异的loxP位点的DNA序列,并进行专一性的剪切和拼接,利用PCR技术将cre基因克隆至原核表达载体pET-29a,在大肠杆菌BL21（DE3）得到了高效表达,采用DEAE－52柱层析的方法对表达蛋白进行了纯化,体外生物学活性检测表明,表达蛋白对含有同向loxP位点的质粒有切割活性。     

Pitx2 deletion in pituitary gonadotropes is compatible with gonadal development, puberty, and fertility

This report introduces a gonadotrope-specific cre transgenic mouse capable of ablating floxed genes in mature pituitary gonadotropes. Initial analysis of this transgenic line, Tg(Lhb-cre)1Sac, reveals that expression is limited to the pituitary cells that produce luteinizing hormone beta, beginning appropriately at e17.5. Cre activity is detectable by a reporter gene in nearly every LHbeta-producing cell, but the remaining hormone-producing cell types and other organs exhibit little to no activity. We used the Tg(Lhb-cre)1Sac strain to assess the role Pitx2 in gonadotrope function. The gonadotrope-specific Pitx2 knockout mice exhibit normal expression of LHbeta, sexual maturation, and fertility, suggesting that Pitx2 is not required for gonadotrope maintenance or for regulated production of gonadotropins.     

Germline recombination in a novel Cre transgenic line,Prl3b1‐Cre mouse

Spermatogenesis is a complex and highly regulated process by which spermatogonial stem cells differentiate into spermatozoa. To better understand the molecular mechanisms of the process, the Cre/loxP system has been widely utilized for conditional gene knockout in mice. In this study, we generated a transgenic mouse line that expresses Cre recombinase under the control of the 2.5 kbp of the Prolactin family 3, subfamily b, member 1 (Prl3b1) gene promoter (Prl3b1‐cre). Prl3b1 was initially reported to code for placental lactogen 2 (PL‐2) protein in placenta along with increased expression toward the end of pregnancy. PL‐2 was found to be expressed in germ cells in the testis, especially in spermatocytes. To analyze the specificity and efficiency of Cre recombinase activity in Prl3b1‐cre mice, the mice were mated with reporter R26GRR mice, which express GFP ubiquitously before and tdsRed exclusively after Cre recombination. The systemic examination of Prl3b1‐cre;R26GRR mice revealed that tdsRed‐positive cells were detected only in the testis and epididymis. Fluorescence imaging of Prl3b1‐cre;R26GRR testes suggested that Cre‐mediated recombination took place in the germ cells with approximately 74% efficiency determined by in vitro fertilization. In conclusion, our results suggest that the Prl3b1‐cre mice line provides a unique resource to understand testicular germ‐cell development. genesis 54:389–397, 2016. © 2016 Wiley Periodicals, Inc.     

A transgenic mouse line expressing cre recombinase in pancreatic β-cells

Transgenic mouse lines expressing Cre recombinase in a cell-specific and tissue-specific manner are essential tools for studying gene function and for developing suitable models for human diseases. Here, we used an expression cassette containing the full 5' untranslated region of the porcine insulin gene to generate a mouse line expressing Cre recombinase specifically in pancreatic β-cells by pronuclear DNA microinjection. We obtained a founder animal that transmitted the construct to its descendants in a Mendelian fashion and whose descendants showed a clear activation of β-galactosidase expression in pancreatic β-cells after crossing into the ROSA26 lacZ reporter mouse line. Cre expression in other organs was negative except for the kidney, intestine, and the cerebral pons where β-galactosidase activity was detected in a small percentage of the cells. This new mouse line is a valuable tool for recombination of floxed alleles in pancreatic β-cells in vivo.     

Cre recombinase activity specific to postnatal, premeiotic male germ cells in transgenic mice

We have generated a transgenic mouse line,Tg(Stra8-cre)1Reb (Stra8-cre), which expresses improved Cre recombinase under the control of a 1.4 Kb promoter region of the germ cell-specific stimulated by retinoic acid gene 8 (Stra8). cre is expressed only in males beginning at postnatal day (P)3 in early-stage spermatogonia and is detected through preleptotene-stage spermatocytes. To further define when cre becomes active, we crossed Stra8-cre males with Tg(ACTB-Bgeo/GFP)21Lbe (Z/EG) reporter females and compared the expression of enhanced green fluorescent protein (EGFP) with the protein encoded by the zinc finger and BTB domain containing 16 (Zbtb16) gene, PLZF-a marker for undifferentiated spermatogonia. Co-expression of EGFP is observed in the majority of PLZF+ cells. We also tested recombination efficiency by mating Stra8-cre;Z/EG males and females with wild-type mice and examining EGFP expression in the offspring. Recombination is detected in >95% of Z/EG+ pups born to Stra8-cre;Z/EG fathers but in none of the offspring born to transgenic mothers, a verification that cre is not functional in females. The postnatal, premeiotic, male germ cell-specific activity of Stra8-cre makes this mouse line a unique resource to study testicular germ cell development.     

消化道细胞表达Cre重组酶转基因小鼠的功能鉴定

目的:检测白蛋白启动子介导的Cre重组酶转基因小鼠Alb-Cre-2中Cre重组酶的组织分布及其在体内介导基因重组的作用。方法:将Alb-Cre小鼠与Smad4条件基因打靶小鼠交配,利用PCR对Cre重组酶介导重组的组织特异性进行检测;然后,将Alb-Cre-2转基因小鼠与ROSA26报告小鼠交配,利用LacZ染色对双转基因阳性子代小鼠进行检测。结果:PCR结果显示心、肺、胰、脑及消化道中Cre重组酶介导的Smad4基因发生重组;LacZ染色进一步表明Cre重组酶在肝细胞、胃壁细胞、空肠潘氏细胞、回肠杯状细胞、大肠杯状细胞、大肠柱状细胞及空泡细胞中特异性表达,并介导ROSA位点LoxP序列间的重组。结论:Alb-Cre-2转基因小鼠在消化道中具有一定的组织特异性,只在胃壁细胞、空肠潘氏细胞、回肠杯状细胞、大肠杯状细胞,大肠柱状细胞及空泡细胞等细胞类型中特异性表达,并能在体内成功地介导这些消化道上皮细胞基因组上LoxP位点间的重组,是一种研制在消化道特定细胞中特异性基因剔除小鼠的良好工具小鼠。     

Cre recombinase specificity defined by the tau locus

We generated a transgenic mouse line (tau::Cre) by targeting the Cre to the tau locus (Mapt). Based on previous reports on the expression of Tau during development, we expected the Cre recombinase to be expressed in a neuron-specific and pan-neuronal manner. However, intercrosses between the tau::Cre and the Cre-activatable reporter animals resulted in offspring with recombination either restricted to the nervous system or throughout the entire conceptus, indicating expression of Tau early in development. The percentage of neuron-specific excision was dependent on the Cre reporter used representing different Cre target sites in the mouse genome. In spite of the observed variability, our data suggest that the tau::Cre mouse line can be used for pan-neuronal recombination of floxed alleles when it is used with caution.     

Inducible Cx40-Cre expression in the cardiac conduction system and arterial endothelial cells

The Connexin-40 (Cx40) gene encodes a gap junction protein that plays an important role in cell-cell communication in cardiomyocytes of the atria and cardiac conduction system and endothelial cells of large arteries. During embryonic development, Cx40 expression is tightly regulated and correlates with progressive ventricular conduction system (VCS) differentiation and vessel function. We have generated Cx40(Cre) mice carrying a CreERT2-IRESmRFP cassette by targeted recombination. In Cx40(Cre) mice, the pattern of expression of RFP is identical to that of the endogenous Cx40 gene and a Cx40(GFP) allele. Using a LacZ-based Cre reporter mouse line, tamoxifen dependent Cre recombination was observed throughout the spatio-temporal profile of Cx40 expression in the VCS and arterial endothelial cells. Cx40(Cre) mice can therefore be used to direct inducible genetic modification in Cx40 expressing cells.