Development of a steroidogenic factor 1/Cre transgenic mouse line

The Cre-loxP strategy provides an approach to disrupt genes in specific tissues and/or cell types, circumventing lethality associated with global knockouts or secondary effects due to gene inactivation at other sites. A critical component is the development of transgenes that target Cre expression to specific cell types. Here, we describe the use of bacterial artificial chromosome (BAC) transgenesis to target Cre expression to tissues that express steroidogenic factor 1 (SF-1, officially designated Nr5a1). Consistent with the SF-1 expression pattern, the SF-1 BAC directed Cre expression to the somatic cells of the gonads, the adrenal cortex, the anterior pituitary, the spleen, and the ventromedial hypothalamic nucleus. This transgene provides a powerful tool to inactivate genes of interest in these tissues.     

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.     

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。     

A CamKIIα iCre BAC allows brain‐specific gene inactivation

Summary: We describe the generation of transgenic mouse lines expressing the Cre recombinase enzyme in brain under control of the CamKIIα gene present in a BAC expression vector. The CamKIIα BAC transgene gave a faithful expression pattern resembling the pattern of the endogenous CamKIIα gene. Specifically, high levels of CamKIIα Cre were detected in hippocampus, cortex, and amygdala, and lower levels were detected in striatum, thalamus, and hypothalamus. As expected, no expression was detected in the cerebellum or outside of the brain. The expression level of the BAC CamKIIα driven Cre was shown to be copy number dependent. To test the activity of the Cre recombinase, the transgenic mice were crossed with mice harbouring the CREB (cAMP response element binding protein) allele with the 10th exon flanked by two loxP sites, and recombination was monitored by the disappearance of the CREB protein. Finally, evaluation of the developmental postnatal expression of the CamKIIα Cre BAC revealed the expression of the Cre recombinase as early as P3. genesis 31:37–42, 2001. © 2001 Wiley‐Liss, Inc.     

Cerebellar granule cell Cre recombinase expression

The cerebellum maintains balance and orientation, refines motor action, stores motor memories, and contributes to the timing aspects of cognition. We generated two mouse lines for making Cre recombinase-mediated gene disruptions largely confined to adult cerebellar granule cells. For this purpose we chose the GABA(A) receptor alpha6 subunit gene, whose expression marks this cell type. Here we describe mouse lines expressing Cre recombinase generated by 1) Cre knocked into the native alpha6 subunit gene by homologous recombination in embryonic stem cells; and 2) Cre recombined into an alpha6 subunit gene carried on a bacterial artificial chromosome (BAC) genomic clone. The fidelity of Cre expression was tested by crossing the mouse lines with the ROSA26 reporter mice. The particular alpha6BAC clone we identified will be valuable for delivering other gene products to cerebellar granule cells.     

Generation and characterization of alpha-chymase-Cre transgenic mice

The Cre-loxP technology allows the introduction of somatic gene alterations in a tissue and/or cell type specific manner. The development of transgenes that target Cre expression to specific cell types is a critical component in this system. Here, we describe the generation and characterization of transgenic mouse lines expressing Cre recombinase under the control of the baboon alpha-chymase promoter, designated Chm:Cre, in order to direct Cre expression specifically to mouse mast cells. Chm:Cre expression was detected in mast cells in lung and colon tissue. Cre-mediated recombination in these mice identified a population of mature tissue resident mast cells using ROSA26R reporter mice. No Cre-expression and Cre-mediated recombination was induced in in vitro generated bone marrow derived mast cells or mast cells isolated from the peritoneal cavity indicating that Cre-expression under the control of the alpha-chymase promoter is solely activated in tissue resident mast cells. These Chm:Cre transgenic mice represent a useful tool to specifically inactivate genes of interest in mast cells of these tissues.     

A Chrnb3‐Cre BAC transgenic mouse line for manipulation of gene expression in retinal ganglion cells

The mechanisms by which retinal ganglion cells (RGCs) make specific connections during development is an intense area of research and have served as a model for understanding the general principles of circuit wiring. As such, genetic tools allowing for specific recombination in RGCs are critical to further our understanding of the cell‐specific roles of different genes during these processes. However, many RGC‐specific Cre lines have drawbacks, due to their broad expression in other cell types and/or retinorecipient regions or lack of expression in broad swaths of the retina. Here, we characterize a Cre BAC transgenic line driven by elements of the cholinergic receptor nicotinic beta 3 subunit (Chrnb3). We show that Cre expression is restricted to RGCs in the retina and sparsely expressed in the brain, importantly excluding retinorecipient regions. Furthermore, Chrnb3‐Cre mice label a wide variety of RGCs distributed throughout the retina and Cre activity is detected embryonically, shortly following RGC differentiation. Finally, we find that Chrnb3‐Cre‐labeled RGCs innervate multiple retinorecipient areas that serve both image‐forming and nonimage forming functions. Thus, this genetic tool will be of broad use to investigators studying the RGC‐specific contributions of genes to visual circuit development.     

Lipid rafts control P2X3 receptor distribution and function in trigeminal sensory neurons of a transgenic migraine mouse model

Background

Tissue-specific gene deletion has proved informative in the analysis of pain pathways. Advillin has been shown to be a pan-neuronal marker of spinal and cranial sensory ganglia. We generated BAC transgenic mice using the Advillin promoter to drive a tamoxifen-inducible CreERT2 recombinase construct in order to be able to delete genes in adult animals. We used a floxed stop ROSA26LacZ reporter mouse to examine functional Cre expression, and analysed the behaviour of mice expressing Cre recombinase.

Results

We used recombineering to introduce a CreERT2 cassette in place of exon 2 of the Advillin gene into a BAC clone (RPCI23-424F19) containing the 5' region of the Advillin gene. Transgenic mice were generated using pronuclear injection. The resulting AvCreERT2 transgenic mice showed a highly specific expression pattern of Cre activity after tamoxifen induction. Recombinase activity was confined to sensory neurons and no expression was found in other organs. Less than 1% of neurons showed Cre expression in the absence of tamoxifen treatment. Five-day intraperitoneal treatment with tamoxifen (2 mg per day) induced Cre recombination events in ≈90% of neurons in dorsal root and cranial ganglia. Cell counts of dorsal root ganglia (DRG) from transgenic animals with or without tamoxifen treatment showed no neuronal cell loss. Sensory neurons in culture showed ≈70% induction after 3 days treatment with tamoxifen. Behavioural tests showed no differences between wildtype, AvCreERT2 and tamoxifen-treated animals in terms of motor function, responses to light touch and noxious pressure, thermal thresholds as well as responses to inflammatory agents.

Conclusions

Our results suggest that the inducible pan-DRG AvCreERT2 deleter mouse strain is a useful tool for studying the role of individual genes in adult sensory neuron function. The pain phenotype of the Cre-induced animal is normal; therefore any alterations in pain processing can be unambiguously attributed to loss of the targeted gene.     

Targeted expression of Cre recombinase to adipose tissue of transgenic mice directs adipose-specific excision of loxP-flanked gene segments.

Functional analysis of mammalian genes relies, in part, on targeted mutations generated by homologous recombination in mice. We have developed a strategy for adipose-specific inactivation of loxP-floxed gene segments. Transgenic mice have been established that express Cre recombinase under the control of the adipose-specific aP2 enhancer/promoter. Crossing of the aP2/ Cre mice with any loxP-floxed gene will facilitate its functional analysis in adipose tissue.     

Generation of BAF53b‐Cre transgenic mice with pan‐neuronal Cre activities

Molecular and functional studies of genes in neurons in mouse models require neuron‐specific Cre lines. The current available neuronal Cre transgenic or knock‐in lines either result in expression in a subset of neurons or expression in both neuronal and non‐neuronal tissues. Previously we identified BAF53b as a neuron‐specific subunit of the chromatin remodeling BAF complexes. Using a bacteria artificial chromosome (BAC) construct containing the BAF53b gene, we generated a Cre transgenic mouse under the control of BAF53b regulatory elements. Like the endogenous BAF53b gene, we showed that BAF53b‐Cre is largely neuron‐specific. In both central and peripheral nervous systems, it was expressed in all developing neurons examined and was not observed in neural progenitors or glial cells. In addition, BAF53b‐Cre functioned in primary cultures in a pan‐neuron‐specific manner. Thus, BAF53b‐Cre mice will be a useful genetic tool to manipulate gene expression in developing neurons for molecular, biochemical, and functional studies. genesis, 53:440–448, 2015. © 2015 Wiley Periodicals, Inc.     

特异性检测胰腺组织表达Cre重组酶转基因小鼠的方法

利用组织特异性分子标志物启动子调控Cre重组酶,研制了6种在不同组织中特异性表达Cre重组酶的转基因小鼠.这些转基因小鼠的基因型鉴定均使用设计在Cre基因编码区的通用引物.为了特异性检测胰腺组织表达Cre重组酶的转基因小鼠,在大鼠胰岛素RIP启动子上和Cre基因上设计1对引物进行PCR扩增,并通过凝胶电泳进行分析.PCR结果显示,设计在Cre基因上的通用引物可以从6种不同组织特异性Cre重组酶转基因小鼠基因组DNA中扩增获得480 bp产物;利用本研究设计的特异性引物可以从胰腺组织表达Cre重组酶转基因小鼠基因组DNA中扩增200 bp的目的条带.这一结果表明,利用特异性引物进行PCR反应,可有效地将胰腺组织表达Cre重组酶转基因小鼠与其他多种组织的Cm重组酶转基因小鼠鉴别开来.     

Efficient BLG-Cre Mediated Gene Deletion in the Mammary Gland

Using the phage P1-derived Cre/loxP recombination system, we have developed a strategy for efficient mammary tissue specific inactivation of floxed genes. Transgenic mice were generated which express Cre DNA-recombinase under the control of the mammary gland specific promoter of the ovine beta- lactoglobulin (BLG) gene. To test the specificity of Cre mediated recombination, we crossed these mice to animals harbouring a floxed DNA ligase I allele. We show that the BLG-Cre construct specifies mammary specific gene deletion, and furthermore that it is temporally regulated, predominantly occurring during lactation. We fully characterised the extent of gene deletion in one line (line 74). In this strain the virgin gland is characterised by low levels (7%) of Cre mediated deletion, whereas 70–80% of cells within the lactating mammary gland have undergone recombination. Immunohisto-chemistry and indirect in situ PCR were used respectively to demonstrate that both Cre protein and Cre activity were evenly distributed throughout the population of secretory epithelial cells. The level of background recombination in non-mammary tissues was found to be 1.1%, irrespective of mammary gland developmental status. Crossing the transgenic BLG-Cre strain described here to mice harbouring other floxed alleles will facilitate the functional analysis of those genes during differentiation and development of the mammary gland.     

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。     

A Probasin‐MerCreMer BAC allows inducible recombination in the mouse prostate

Tissue‐specific transgene expression in the prostate epithelium has previously been achieved using short prostate‐specific promoters, rendering transgenic mouse lines susceptible to integration site‐dependent effects. Here we demonstrate the applicability of bacterial artificial chromosome (BAC) technology to transgene expression in the prostate epithelium. We present mouse lines expressing an inducible Cre protein (MerCreMer) under the control of regulatory elements of the probasin gene on a BAC. These mouse lines show high organ specificity, high transgene expression in anterior, dorsal and lateral prostate lobes, no background Cre recombination using a reporter strain and adjustable amounts of Cre‐induced recombination upon tamoxifen induction. Together with two recently reported transgenic lines expressing the Cre‐ERT2 protein from small prostate‐specific promoters, these mouse lines will be useful in research focused on prostate‐specific disorders such as benign hyperplasia or cancer. genesis 47:757–764, 2009. © 2009 Wiley‐Liss, Inc.     

Odontoblast-specific expression of cre recombinase successfully deletes gene segments flanked by loxP sites in mouse teeth

Embryonic or neonatal lethality of mice with targeted disruption of critical genes preclude them from further characterization of specific roles of these genes during postnatal development and aging. In order to study the molecular roles of such genes in teeth, we generated transgenic mouse lines expressing bacteriophage Cre recombinase under the control of the mouse dentin sialophosphoprotein (dspp) gene promoter. The expression of Cre recombinase protein was mainly detected in the nucleus of the odontoblasts. The efficiency of Cre activity was analyzed by crossing the Dspp-Cre mice with ROSA26 reporter (R26R) mice. The offspring with both genotypes have shown specific deletion of intervening sequences flanked by loxP sites upstream of the reporter gene, thereby facilitating the expression of the beta-galactosidase (beta-gal) gene in the teeth. The activity of beta-gal was initially observed in the odontoblasts of 1-day-old mice and increased with tooth development. Almost all of the odontoblasts have shown lacZ activity by 3 weeks of age. We could not detect Cre recombinase activity in any other cells, including ameloblasts. These studies indicate that the Dspp-Cre transgenic mice will be valuable to generate odontoblast-specific gene knockout mice so as to gain insight into the molecular roles of critical genes in the odontoblasts during dentinogenesis.     

Cre-mediated gene deletion in the mammary gland.

To delete genes specifically from mammary tissue using the Cre-lox system, we have established transgenic mice expressing Cre recombinase under control of the WAP gene promoter and the MMTV LTR. Cre activity in these mice was evaluated by three criteria. First, the tissue distribution of Cre mRNA was analyzed. Second, an adenovirus carrying a reporter gene was used to determine expression at the level of single cells. Third, tissue specificity of Cre activity was determined in a mouse strain carrying a reporter gene. In adult MMTV-Cre mice expression of the transgene was confined to striated ductal cells of the salivary gland and mammary epithelial cells in virgin and lactating mice. Expression of WAP-Cre was only detected in alveolar epithelial cells of mammary tissue during lactation. Analysis of transgenic mice carrying both the MMTV-Cre and the reporter transgenes revealed recombination in every tissue. In contrast, recombination mediated by Cre under control of the WAP gene promoter was largely restricted to the mammary gland but occasionally observed in the brain. These results show that transgenic mice with WAP-Cre but not MMTV-Cre can be used as a powerful tool to study gene function in development and tumorigenesis in the mammary gland.     

胰腺组织表达Cre重组酶转基因小鼠的建立及鉴定

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基因剔除研究的重要工具。为了建立胰腺组织特异性Cre转基因小鼠,我们通过PCR克隆了大鼠胰岛素基因启动子,并用它指导Cre基因在胰岛细胞中的特异性表达。在Cre重组酶基因5′端添加了真核核糖体结合序列和核定位序列以使Cre重组酶能穿越核膜在细胞核中发挥功能;同时,在Cre基因3′端添加了含内含子的3′端人生长激素基因。表达载体经显微注射导入小鼠受精卵以建立转基因小鼠。PCR检测显示共获得7只Cre整合阳性的转基因首建者小鼠;RTPCR结果表明其中1只首建者小鼠的子代鼠在胰腺中转录了外源基因,进一步的Southern杂交结果表明,该转基因小鼠能够在胰腺中表达有功能的Cre重组酶。      

A morpholino phenocopy of the cyclops mutation

Summary: Conditional and tissue specific gene targeting using the Cre‐loxP recombination system in combination with established ES cell techniques has become a standard for in vivo loss of function studies. In a typical flox and delete gene targeting strategy, the loxP‐neo‐loxP cassette is inserted into an intron and an additional loxP site is located in one of the homology arms so that loxP sites surround a functionally essential part of the gene. The neo cassette in usually removed by transient expression of the Cre recombinase in ES cells to avoid selection gene interference and genetic ambiquity. However, this causes a significant increase in manipulation of ES cells and often compromises ES cell pluripotency. Here we describe a method in which the floxed neo gene is removed from a knockout allele by infecting 16‐cell‐stage morulae by the recombinant Cre adenovirus. This virus provides only transient Cre expression and does not integrate into the mouse genome. Produced mosaic mice transmitted the desired allele without the neo cassette with high frequency to their offspring. This method is rapid and easy and does not require any special equipment. Moreover, because superovulated mice can be used as donors, this method does not necessitate a large number of mice. genesis 31:126–129, 2001. © 2001 Wiley‐Liss, Inc.     

Selective removal of the selenocysteine tRNA [Ser]Sec gene (Trsp) in mouse mammary epithelium

Mice homozygous for an allele encoding the selenocysteine (Sec) tRNA [Ser]Sec gene (Trsp) flanked by loxP sites were generated. Cre recombinase-dependent removal of Trsp in these mice was lethal to embryos. To investigate the role of Trsp in mouse mammary epithelium, we deleted this gene by using transgenic mice carrying the Cre recombinase gene under control of the mouse mammary tumor virus (MMTV) long terminal repeat or the whey acidic protein promoter. While both promoters target Cre gene expression to mammary epithelium, MMTV-Cre is also expressed in spleen and skin. Sec tRNA [Ser]Sec amounts were reduced by more than 70% in mammary tissue with either transgene, while in skin and spleen, levels were reduced only with MMTV-Cre. The selenoprotein population was selectively affected with MMTV-Cre in breast and skin but not in the control tissue, kidney. Moreover, within affected tissues, expression of specific selenoproteins was regulated differently and often in a contrasting manner, with levels of Sep15 and the glutathione peroxidases GPx1 and GPx4 being substantially reduced. Expression of the tumor suppressor genes BRCA1 and p53 was also altered in a contrasting manner in MMTV-Cre mice, suggesting greater susceptibility to cancer and/or increased cell apoptosis. Thus, the conditional Trsp knockout mouse allows tissue-specific manipulation of Sec tRNA and selenoprotein expression, suggesting that this approach will provide a useful tool for studying the role of selenoproteins in health.