共查询到20条相似文献,搜索用时 0 毫秒
1.
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。 相似文献
2.
Hidemi Misawa Daijiro Inomata Miseri Kikuchi Sae Maruyama Yasuhiro Moriwaki Takashi Okuda Nobuyuki Nukina Tomoyuki Yamanaka 《Genesis (New York, N.Y. : 2000)》2016,54(11):568-572
VAChT‐Cre.Fast and VAChT‐Cre.Slow mice selectively express Cre recombinase in approximately one half of postnatal somatic motor neurons. The mouse lines have been used in various studies with selective genetic modifications in adult motor neurons. In the present study, we crossed VAChT‐Cre lines with a reporter line, CAG‐Syp/tdTomato, in which synaptophysin‐tdTomato fusion proteins are efficiently sorted to axon terminals, making it possible to label both cell bodies and axon terminals of motor neurons. In the mice, Syp/tdTomato fluorescence preferentially co‐localized with osteopontin, a recently discovered motor neuron marker for slow‐twitch fatigue‐resistant (S) and fast‐twitch fatigue‐resistant (FR) types. The fluorescence did not preferentially co‐localize with matrix metalloproteinase‐9, a marker for fast‐twitch fatigable (FF) motor neurons. In the neuromuscular junctions, Syp/tdTomato fluorescence was detected mainly in motor nerve terminals that innervate type I or IIa muscle fibers. These results suggest that the VAChT‐Cre lines are Cre‐drivers that have selectivity in S and FR motor neurons. In order to avoid confusion, we have changed the mouse line names from VAChT‐Cre.Fast and VAChT‐Cre.Slow to VAChT‐Cre.Early and VAChT‐Cre.Late, respectively. The mouse lines will be useful tools to study slow‐type motor neurons, in relation to physiology and pathology. 相似文献
3.
肾上腺是人体重要的内分泌器官。由于缺乏肾上腺皮质束状带特异性表达Cre酶的工具鼠,目前对肾上腺皮质束状带细胞中特异表达基因的功能缺乏深入的解析。CYP11B1基因编码类固醇11β-羟化酶,该酶是糖皮质激素合成的关键酶,在肾上腺皮质束状带中特异性表达。本研究旨在利用CYP11B1基因在束状带特异性表达的特点,构建在肾上腺皮质束状带中特异性表达Cre重组酶的转基因动物。采用CRISPR/Cas9技术在CYP11B1基因终止密码子位点定点敲入2A-GfpCre表达框,获得CYP11B1-2A-GfpCre同源重组载体,进而构建CYP11B1Cre小鼠,并通过mTmG和LacZ染色确定Cre酶主要表达在小鼠肾上腺皮质束状带。在此基础上,本研究还用该工具鼠与胱硫醚-γ-裂解酶(cystathionineγ-lyase, CTH)条件性敲除鼠交配,获得了肾上腺皮质束状带CTH特异性敲除的小鼠,并证实了该动物肾上腺皮质束状带中CTH表达缺失。以上结果充分说明肾上腺皮质束状带特异性表达Cre重组酶小鼠构建成功。该工具鼠的成功构建,为深入研究肾上腺皮质束状带相关功能提供了有力工具。 相似文献
4.
Zhang XM Ng AH Tanner JA Wu WT Copeland NG Jenkins NA Huang JD 《Genesis (New York, N.Y. : 2000)》2004,40(1):45-51
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. 相似文献
5.
Generation of a transgenic mouse model with chondrocyte-specific and tamoxifen-inducible expression of Cre recombinase 总被引:2,自引:0,他引:2
Chen M Lichtler AC Sheu TJ Xie C Zhang X O'Keefe RJ Chen D 《Genesis (New York, N.Y. : 2000)》2007,45(1):44-50
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. 相似文献
6.
We use a transgenic mouse model system to elucidate the regulatory regions within the human cholinergic gene locus responsible for vesicular acetylcholine transporter gene expression in vivo. In this report we characterized two transgenes for their ability to confer cholinergic-specific expression of the encoded vesicular acetylcholine transporter. An 11.2 kb transgene (named hV11.2) that spanned from about 5 kb upstream of the start of vesicular acetylcholine transporter translation down to the first choline acetyltransferase coding exon gave expression in the somatomotor neurons and a subpopulation of cholinergic neurons in the medial habenular nucleus. The second transgene (named hV6.7), a 5-prime truncated version of hV11.2 that was devoid of 4.5 kb of gene-regulatory sequences completely lacked vesicular acetylcholine transporter expression in vivo. Our data indicate that vesicular acetylcholine transporter expression in somatomotor neurons and in the medial habenular nucleus is uniquely specified within the cholinergic gene locus, and separable from cholinergic expression elsewhere. The identification of these two subdivisions of the cholinergic nervous system suggests that other cholinergic neurons in the CNS and PNS are similarly regulated by additional discrete domains within the cholinergic gene locus. 相似文献
7.
Zachary E. Stine Jimmy L. Huynh Stacie K. Loftus David U. Gorkin Amirali H. Salmasi Thomas Novak Todd Purves Ronald A. Miller Anthony Antonellis John P. Gearhart William J. Pavan Andrew S. McCallion 《Genesis (New York, N.Y. : 2000)》2009,47(11):765-770
Utilizing a recently identified Sox10 distal enhancer directing Cre expression, we report S4F:Cre, a transgenic mouse line capable of inducing recombination in oligodendroglia and all examined neural crest derived tissues. Assayed using R26R:LacZ reporter mice expression was detected in neural crest derived tissues including the forming facial skeleton, dorsal root ganglia, sympathetic ganglia, enteric nervous system, aortae, and melanoblasts, consistent with Sox10 expression. LacZ reporter expression was also detected in non‐neural crest derived tissues including the oligodendrocytes and the ventral neural tube. This line provides appreciable differences in Cre expression pattern from other transgenic mouse lines that mark neural crest populations, including additional populations defined by the expression of other SoxE proteins. The S4F:Cre transgenic line will thus serve as a powerful tool for lineage tracing, gene function characterization, and genome manipulation in these populations. genesis 47:765–770, 2009. © 2009 Wiley‐Liss, Inc. 相似文献
8.
Parietal cells are one of the largest epithelium cells of the mucous membrane of the stomach that secrete hydrochloric acid.To study the function of gastric parietal cells during gastric epithelium homeostasis,we generated a transgenie mouse line,namely,Atp4b-Cre,in which the expression of Cre recombinase was controlled by a 1.0 kb promoter of mouse β-subunit of H+-,K+-ATPase gene(Atp4b).In order to test the tissue distribution and excision activity of Cre recombinase in vivo,the Atp4b-Cre transgenic mice were bred with the reporter strain ROSA26 and a mouse strain that carries Smad4 conditional alleles(Smad4Co/Co).Multiple-tissue PCR of Atp4b-Cre;Smad4Co/+mice revealed that the recombination only happened in the stomach.As indicated by LacZ staining,ROSA26;Atp4b-Cre double transgenic mice showed efficient expression of Cre recombinase within the gastric parietal cells.These results showed that this Atp4b-Cre mouse line could be used as a powerful tool to achieve conditional gene knockout in gastric parietal cells. 相似文献
9.
10.
Targeted expression of Cre recombinase in macrophages and osteoclasts in transgenic mice 总被引:3,自引:0,他引:3
To develop specific conditional gene ablation in the hematopoietic myeloid-osteoclast lineage, transgenic mice expressing Cre recombinase under the control of the CD11b promotor were generated on the C57BL/6 background. The cellular specificity of Cre activity following recombination was quantified in the Z/EG reporter transgenic mice by FACS analysis with lineage-specific markers and EGFP coexpression. A high degree of recombination, as evidenced by EGFP-positive cells, was demonstrated in macrophages and granulocytes of bone marrow and spleen by the presence of double-positive cells CD11b/EGFP and Gr1/EGFP, respectively. Interestingly, the peritoneal macrophage population showed almost complete DNA recombination at large. Most important, mature osteoclast cells derived from the double transgenic bone marrow and spleen progenitors were EGFP-positive. Hence, these CD11b-Cre mice will provide a unique tool to unravel novel gene function and activities involved during osteoclast and macrophage differentiation and maturation processes. 相似文献
11.
Zengming Zhao Yanxun Sun Ning Hou Yan Teng Youliang Wang Xiao Yang 《Genesis (New York, N.Y. : 2000)》2009,47(10):674-679
Gastric pit cells are high‐turnover epithelial cells of the gastric mucosa. They secrete mucus to protect the gastric epithelium from acid and pepsin. To investigate the genetic mechanisms underlying the physiological functions of gastric pit cells, we generated a transgenic mouse line, namely, Capn8‐Cre, in which the expression of Cre recombinase was controlled by the promoter of the intracellular Ca2+‐regulated cysteine protease calpain‐8. To test the tissue distribution and excision activity of Cre recombinase, the Capn8‐Cre transgenic mice were bred with the ROSA26 reporter strain and a mouse strain that carries Smad4 conditional alleles (Smad4Co/Co). Multiple‐tissue PCR and LacZ staining demonstrated that Capn8‐Cre transgenic mouse expressed Cre recombinase in the gastric pit cells. Cre recombinase activity was also detected in the liver and skin tissues. These data suggest that the Capn8‐Cre mouse line described here could be used to dissect gene function in gastric pit cells. genesis 47:674–679, 2009. © 2009 Wiley‐Liss, Inc. 相似文献
12.
Dlx5 and Dlx6, two members of the Distalless gene family, are required for development of numerous tissues during embryogenesis, including facial and limb development. This gene pair is expressed in tandem, transcribed toward each other and separated by a short intergenic region containing multiple putative enhancers. Targeted inactivation of Dlx5 and Dlx6 in mice results in multiple developmental defects in craniofacial and limb structures, suggesting that these genes are crucial for aspects of both neural crest and nonneural crest development. To further investigate potential developmental roles of Dlx5 and Dlx6, we used one of the Dlx5/6 intergenic enhancers to drive Cre recombinase expression in transgenic mice. Crossing Dlx5/6-Cre transgenic mice with mice from the R26R strain results in beta-galactosidase staining in the apical ectodermal ridge, brain, and neural crest-derived mesenchyme of the pharyngeal arches, with staining in term embryos observed in the facial skeleton and specific brain structures. However, in contrast to endogenous expression patterns of Dlx5 and Dlx6, Cre expression within the pharyngeal arches occurs during a very narrow window in early development. Our studies suggest that Dlx5/6-Cre mice may prove useful both in further understanding the function and regulation of Distalless genes during development and in studies of gene function in conditional knockout mice. 相似文献
13.
Several lines of evidence indicate that nerve growth factor is important for the development and maintenance of the basal forebrain cholinergic phenotype. In the present study, using rat primary embryonic basal forebrain cultures, we demonstrate the differential regulation of functional cholinergic markers by nerve growth factor treatment (24–96 h). Following a 96‐h treatment, nerve growth factor (1–100 ng/mL) increased choline acetyltransferase activity (168–339% of control), acetylcholine content (141–185%), as well as constitutive (148–283%) and K+‐stimulated (162–399%) acetylcholine release, but increased release was not accompanied by increased high‐affinity choline uptake. Enhancement of ACh release was attenuated by vesamicol (1 µm ), suggesting a vesicular source, and was abolished under choline‐free conditions, emphasizing the importance of extracellular choline as the primary source for acetylcholine synthesized for release. A greater proportion of acetylcholine released from nerve growth factor‐treated cultures than from nerve growth factor‐naïve cultures was blocked by voltage‐gated Ca2+ channel antagonists, suggesting that nerve growth factor modified this parameter of neurotransmitter release. Cotreatment of NGF (20 ng/mL) with K252a (200 nm ) abolished increases in ChAT activity and prevented enhancement of K+‐stimulated ACh release beyond the level associated with K252a, suggesting the involvement of TrkA receptor signaling. Also, neurotrophin‐3, neurotrophin‐4 and brain‐derived neurotrophic factor (all at 5–200 ng/mL) increased acetylcholine release, although they were not as potent as nerve growth factor and higher concentrations were required. High brain‐derived neurotrophic factor concentrations (100 and 200 ng/mL) did, however, increase release to a level similar to nerve growth factor. In summary, long‐term exposure (days) of basal forebrain cholinergic neurons to nerve growth factor, and in a less‐potent fashion the other neurotrophins, enhanced the release of acetylcholine, which was dependent upon a vesicular pool and the availability of extracellular choline. 相似文献
14.
Sarah C. Tryon Iris M. Sakamoto Kris F. Kaigler Gabriella Gee Jarrett Turner Katherine Bartley Jim R. Fadel Marlene A. Wilson 《Genes, Brain & Behavior》2023,22(1):e12837
The cholinergic system is a critical regulator of Pavlovian fear learning and extinction. As such, we have begun investigating the cholinergic system's involvement in individual differences in cued fear extinction using a transgenic ChAT::Cre rat model. The current study extends behavioral phenotyping of a transgenic ChAT::Cre rat line by examining both freezing behavior and ultrasonic vocalizations (USVs) during a Pavlovian cued fear learning and extinction paradigm. Freezing, 22 kHz USVs, and 50 kHz USVs were compared between male and female transgenic ChAT::Cre+ rats and their wildtype (Cre-) littermates during fear learning, contextual and cue-conditioned fear recall, cued fear extinction, and generalization to a novel tone. During contextual and cued fear recall ChAT::Cre+ rats froze slightly more than their Cre- littermates, and displayed significant sex differences in contextual and cue-conditioned freezing, 22 kHz USVs, and 50 kHz USVs. Females showed more freezing than males in fear recall trials, but fewer 22 kHz distress calls during fear learning and recall. Females also produced more 50 kHz USVs during exposure to the testing chambers prior to tone (or shock) presentation compared with males, but this effect was blunted in ChAT::Cre+ females. Corroborating previous studies, ChAT::Cre+ transgenic rats overexpressed vesicular acetylcholine transporter immunolabeling in basal forebrain, striatum, basolateral amygdala, and hippocampus, but had similar levels of acetylcholinesterase and numbers of ChAT+ neurons as Cre- rats. This study suggests that variance in behavior between ChAT::Cre+ and wildtype rats is sex dependent and advances theories that distinct neural circuits and processes regulate sexually divergent fear responses. 相似文献
15.
16.
17.
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. 相似文献
18.
19.
The number of transgenic mouse lines expressing Cre in either type of pigment cells (melanocytes and retinal pigment epithelium, RPE) is limited, and the available lines do not always offer sufficient specificity. In this study, we addressed this issue and we report on the generation of a MART‐1::Cre BAC transgenic mouse line, in which the expression of Cre recombinase is controlled by regulatory elements of the pigment cell‐specific gene MART‐1 (mlana). When MART‐1::Cre BAC transgenic mice were bred with the ROSA26‐R reporter line, ß‐galactosidase expression was observed in RPE from E12.5 onwards, and in melanocyte precursors from E17.5, indicating that the MART‐1::Cre line provides Cre recombinase activity in pigment‐producing cells rather than in a particular lineage. In addition, breeding of this mouse line to mice carrying a conditional allele of RBP‐Jκ corroborated the reported phenotypes in both pigment cell lineages, inducing hair greying and microphthalmia. Our results thus suggest, that the MART‐1::Cre line may serve as a novel and useful tool for functional studies in melanocytes and the RPE.genesis 49:403–409, 2011. © 2011 Wiley‐Liss, Inc. 相似文献