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。     

软骨组织特异性表达Cre重组酶转基因小鼠的研制和鉴定

构建了含有软骨组织特异性Ⅱ型胶原A1启动子和Cre重组酶基因的转基因载体pcol2Al-Cre。323枚小鼠受精卵经显微注射引入转基因片段后,分别移植至14只假孕母鼠的输卵管使其发育。共得到仔鼠52只,PCR结果显示其中10只小鼠基因组上有Cre基因的整合,整合率为19．2％。用整合有Cre基因的转基因小鼠与基因组上携带LoxP位点的条件基因打靶小鼠交配,以检测Cre酶介导的重组及其组织特异性。PCR结果表明：col2Al-Cre转基因小鼠软骨组织中表达的Cre重组酶成功地介导了LoxP之间的重组。此结果通过Southern杂交得到了进一步的证实。     

中枢神经系统特异性表达Cre重组酶的转基因小鼠

利用从129sv小鼠基因组文库克隆得到的1．8kb的胶质细胞原纤维酸性蛋白(GFAP)基因的5′端调控序列,构建了含有2个β—珠蛋白绝缘子、GFAP5′端调控区、Cre基因和人生长激素基因(hGH)polyA的转基因载体pGFAP—Cre—hGH。以显微注射的方法将7．6kb的转基因片段pGFAP—Cre—hGH引入191枚小鼠基因组受精卵,其中176枚分别移植至8只假孕母鼠的输卵管中使其发育,共获得子代小鼠25只。经PCR和Southern杂交鉴定其中7只小鼠基因组上整合有Cre基因,整合率为28％。用整合有Cre基因的转基因小鼠与基因组上整合有LoxP位点和LacZ表达框的ROSA26鼠杂交,以检测Cre酶的活性、组织特异性及其介导的两个LoxP位点间的重组。LacZ染色结果表明,GFAP—Cre转基因小鼠只在中枢神经系统中表达Cre重组酶并能在体内成功介导LoxP位点间的重组。     

肥大软骨细胞特异性表达Cre重组酶转基因小鼠的建立

肥大软骨细胞是软骨细胞的终末分化形式,在软骨内成骨过程中发挥十分关键的作用。为了研究肥大软骨细胞在骨骼发育过程中的功能,我们构建了在8.2 kb小鼠X型胶原基因（Col10a1）启动子控制下表达Cre重组酶的转基因小鼠品系（Col10a1-8.2-Cre）。采用显微注射法将11.5 kb的转基因片段引入小鼠基因组,共注射受精卵328枚,获得子代鼠51只,经PCR基因型鉴定有3只在基因组上整合有Cre重组酶基因。PCR检测发现Col10a1-8.2-Cre转基因在含有肥大软骨细胞的组织中表达。为了检测Cre重组酶表达的强度和组织特异性,转基因小鼠与ROSA26报告小鼠交配。子代ROSA26;Col10a1-8.2-Cre双转基因小鼠LacZ染色检测的结果显示,Cre重组酶在所有的肥大软骨细胞中表达。原位杂交的结果验证Col10a1-8.2-Cre转基因表达在肥大区的上端。以上结果表明,我们建立的肥大软骨细胞特异性表达Cre重组酶的转基因小鼠品系可以作为一种遗传学工具,介导目的基因在肥大软骨细胞中的敲除。     

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。     

血管内皮细胞特异表达Cre重组酶转基因小鼠的建立

血管内皮细胞参与血管形成、血管稳态维持、血栓形成、炎症和血管重建等生理和病理过程。为了便于通过Cre-LoxP系统研究相关基因在血管内皮细胞中的功能,创建了Tie2-Cre转基因小鼠,利用Tie2基因的启动子驱动Cre重组酶基因在血管内皮细胞中表达。经基因组PCR和Southern Blot鉴定有6只小鼠在基因组上整合有Cre基因,整合率为11％。为了验证Cre重组酶的剪切活性和表达组织分布,我们将Tie2-Cre转基因小鼠分别与Smad4条件基因打靶小鼠和报告小鼠ROSA26交配。Tie2-Cre;Smad4^co/＋小鼠的多个组织的基因组DNA的PCR结果显示,Cre重组酶在所有包含血管内皮细胞的组织中表达并能介导LoxP间的重组。Tie2-Cre;ROSA26双转基因胚胎LacZ染色结果显示,Cre重组酶在所有被检测组织的血管内皮细胞中特异性表达。因此．Tie2-Cre转基因小鼠可作血管内皮细胞谱系分析和在血管内皮细胞进行条件基因打靶的理想工具小鼠。     

角质细胞特异性表达Cre重组酶转基因小鼠的建立

构建了含有角质细胞特异性角质素5启动子、Cre重组酶基因和人生长激素基因plyA的转基因载体pK5-Cre-hGH。以显微注射的方法将4．2kb的转基因片段K5-Cre-hGH引入小鼠基因组,共注射720枚受精卵,其中龄5枚移植至29只假孕母鼠的输卵管中发育,获得子代小鼠48只,经基因型鉴定有12只小鼠在其基因组上整合有Cre基因,整合率为25％。将带有cre重组酶基因的小鼠与基因组上携带loxP位点的smad4条件基因打靶小鼠杂交以检测Cre重组酶组织特异性表达情况以及介导重组的功能。结果表明,K5-Cre转基因小鼠只在皮肤组织中表达Cre重组酶并能在体内成功地介导loxP位点的重组。     

增强子驱动的Cre转基因小鼠的构建及Cre基因的表达鉴定

目的:探索将增强子应用于构建Cre转基因小鼠品系,为以条件基因敲除为基础的基因功能研究提供更多的工具。方法:通过PCR方法从小鼠的细菌人工染色体扩增UH增强子片段,构建含有Hsp68基础启动子、增强子UH、Cre重组酶基因和SV40 polyA的转基因载体pLW400,将3.3 kb的转基因片段通过显微注射导入小鼠受精卵;为了检测Cre在转基因小鼠中的表达,将转基因一代小鼠与纯合子ROSA26报告小鼠(R/R)交配,收集第14 d胚胎期(E14)的舌组织进行LacZ染色检测鉴定。结果:经鉴定,31只子代小鼠中有6只携带外源基因,整合率为19.4%;与R/+对照相比,E14期的双基因型Cre,R/+舌组织为阳性结果(蓝色)。这表明Cre基因在转基因小鼠舌组织内得到表达,并在体内介导ROSA26基因座loxP位点间的重组,且有效删除了2个loxP之间的片段,从而启动了LacZ基因的表达。结论:构建了UH增强子-Hsp68Cre的转基因小鼠,在舌肌中特异表达Cre基因,提示增强子可以被选择应用于Cre转基因小鼠的构建;为舌肌的发育和再生研究奠定了基础。     

平滑肌细胞特异表达Cre重组酶转基因小鼠Cre重组酶的表达分布

利用转基因技术构建了平滑肌细胞特异表达Cre重组酶的转基因小鼠（SMA-Cre）,将该小鼠与基因组上携带LoxP位点的ROSA26小鼠杂交,通过LacZ染色检测Cre重组酶介导重组的功能以及表达的组织特异性。结果显示,在气管C形软骨连接处平滑肌、细支气管壁平滑肌、食道壁平滑肌、胃壁平滑肌、小肠壁平滑肌、子宫壁平滑肌、膀胱壁平滑肌、血管壁平滑肌、心肌及骨骼肌中检测到Cre重组酶活性。表明SMA—Cre转基因小鼠在所有平滑肌细胞中表达Cre重组酶,并且有很好的组织特异性。     

乳腺上皮细胞特异性敲除Scrib基因杂合子小鼠的制作与鉴定

目的利用Cre．LoxP重组酶系统构建乳腺上皮细胞特异性敲除Serib基因杂合子小鼠,并进行鉴定,为进一步在动物整体水平研究Scrib基因在乳腺癌中的作用提供研究平台。方法将Scrib条件敲除杂合子小鼠（Scrib＋/ft小鼠）进行繁殖并鉴定,然后将鉴定结果为阳性的子代Scrib＋/ft小鼠与乳腺上皮细胞特异性表达Cre重组酶的MMTV．Cre纯合子小鼠进行杂交,鉴定其子代小鼠的基因型。结果成功繁育Scrib条件敲除小鼠和MMTV．Cre小鼠,并通过鉴定得到Scrib＋/ft小鼠,与MMTV-Cre小鼠杂交并繁殖,获得基因型为Scrib＋/ft;MMTVCre＋/-小鼠5只。结论本研究利用Cre．LoxP重组酶系统成功构建了乳腺上皮细胞特异性敲除Scrib基因杂合子小鼠,为进一步研究极性蛋白Scrib表达下调在乳腺癌发生中的作用提供了良好的动物模型。     

Mouse reporter strain for noninvasive bioluminescent imaging of cells that have undergone Cre-mediated recombination

Conditional alleles containing LoxP recombination sites, in conjunction with Cre recombinase delivered by a variety of means, allows for spatial and temporal control of gene expression in mouse models. Here we describe a mouse strain in which a luciferase (Luc) cDNA, preceded by a LoxP-stop-LoxP (L-S-L) cassette, was introduced into the ubiquitously expressed ROSA26 locus. Mouse embryo fibroblasts derived from this strain expressed luciferase after Cre-mediated recombination in vitro. ROSA26 L-S-L-Luc/+ mice expressed luciferase in a diffuse or liver-restricted pattern, as determined by noninvasive, bioluminescent imaging, when crossed to transgenic mice in which Cre was under the control of a zygotically expressed (EIIA-Cre), or a liver-restricted (albumin-Cre), promoter, respectively. Organ-specific luciferase expression was also seen after intraparenchymal administration of an adenovirus encoding Cre. The ROSA26 L-S-L-Luc/+ strain should be useful for characterizing Cre mouse strains and for following the fate of cells that have undergone Cre-mediated recombination in vivo.     

Collagenl α1 promoter drives the expression of Cre recombinase in osteoblasts of transgenic mice

Osteoblasts participate in bone formation,bone mineralization,osteoclast differentiation and many pathological processes.To study the function of genes in osteoblasts using Cre-LoxP system,we generated a mouse line expressing the Cre recombinase under the control of the rat Collagenlal (Coilal) promoter(Coilatl-Cre).Two founders were identified by genomic PCR from 16 offsprings.and the integration efficiency is 12.5%.In order tO determine the tissue distribution and the activity of Cre rccombinase in the transgenic mice,the Collal-Cre transgenic mice were bred with the ROSA26 reporter strain and a mouse strain that carries Smad4 conditional alleles (Smad4co/co).Multiple tissue PCR of Collal-Cre;Smad4co/ mice revealed the restricted Cre activity in bone tissues containing osteoblasts and tendon.LacZ staining in the Coilal-Cre;ROSA26 double transgenic mice revealed that the Cre recombinase began to express in the osteoblasts of calvaria at E14.5.Cre activity was observed in the osteoblasts and osteocytes of P10 double transgenic mice.All these data indicated that the Collal-Cre transgenic mice could Serve as a valuabletool for osteoblast lineage analysis and conditional gene knockout in osteoblasts.     

DNA重组酶Cre介导载体间基因的重组转移

DNA重组酶Cre可以识别LoxP位点,使含有LoxP位点的DNA分子发生重组:2个同向LoxP之间的DNA片段被删除,2个环状DNA分子被整合为一个大分子.基于Cre酶的这些作用特性,构建了一套载体间基因的重组转移体系,在Cre酶的作用下,gfp基因被从基因供体pTLG上切除下来,然后转移到基因受体pET-LoxP上,从而快速、简便地完成了gfp基因高效表达载体pET-gfp的构建.gfp基因在大肠杆菌BL21(DE3)中被诱导表达,使菌落产生了可视的绿色荧光.通过对荧光菌落的计数分析,比较了环状基因供体pTLG和线性基因供体pTLG对有效重组率的影响.使繁琐的传统载体构建变为简单的酶促反应,极大地简化了载体构建步骤,为Cre酶在基因克隆和亚克隆中的应用提供了很好的研究基础.     

Collagen1alpha1 promoter drives the expression of Cre recombinase in osteoblasts of transgenic mice

Osteoblasts participate in bone formation, bone mineralization, osteoclast differentiation and many pathological processes. To study the function of genes in osteoblasts using Cre-LoxP system, we generated a mouse line expressing the Cre recombinase under the control of the rat Collagen1alpha1 (Col1alpha1) promoter (Col1alpha1-Cre). Two founders were identified by genomic PCR from 16 offsprings, and the integration efficiency is 12.5%. In order to determine the tissue distribution and the activity of Cre recombinase in the transgenic mice, the Col1alpha1-Cre transgenic mice were bred with the ROSA26 reporter strain and a mouse strain that carries Smad4 conditional alleles (Smad4(Co/Co)). Multiple tissue PCR of Col1alpha1-Cre;Smad4(Co/+)mice revealed the restricted Cre activity in bone tissues containing osteoblasts and tendon. LacZ staining in the Col1alpha1-Cre;ROSA26 double transgenic mice revealed that the Cre recombinase began to express in the osteoblasts of calvaria at E14.5. Cre activity was observed in the osteoblasts and osteocytes of P10 double transgenic mice. All these data indicated that the Col1alpha1-Cre transgenic mice could serve as a valuable tool for osteoblast lineage analysis and conditional gene knockout in osteoblasts.     

Atp4b promoter directs the expression of Cre recombinase in gastric parietal cells of transgenic mice

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.     

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.     

Transgenic mice that express Cre recombinase in osteoclasts

To study the physiological control of osteoclasts, the bone resorbing cells, we generated transgenic mice carrying the Cre recombinase gene driven by either the tartrate-resistant acid phosphatase (TRAP) or cathepsin K (Ctsk) promoters. TRAP-Cre and Ctsk-Cre transgenic mouse lines were characterized by breeding with LacZ ROSA 26 (R26R) reporter mice and immunohistochemistry for Cre recombinase. The Cre transgene was functional in all lines, with Cre-mediated recombination occurring primarily in the long bones, vertebrae, ribs, and calvaria. Histological analyses of the bones demonstrated that functional Cre protein was present in 1) osteoclasts (Ctsk-Cre); 2) osteoclasts, columnar proliferating, and hypertrophic chondrocytes (TRAP-Cre line 4); and 3) round proliferating chondrocytes (TRAP-Cre line 3). In conclusion, we generated transgenic mouse lines that will enable the deletion of floxed target genes in osteoclasts, which will be valuable tools for studying the regulation of osteoclast function.