Retina-specific gene excision by targeted expression of Cre recombinase

The use of Cre recombinase for conditional targeting permits the controlled removal or activation of genes in specific tissues and at specific times of development. The Rho–Cre mice provide an improved tool for studying gene ablation in rod photoreceptor cells. To establish a robust expression of Rho–Cre transgenic mice that would be useful for the study of various protein functions in photoreceptor cells, a total 11,987 kb fragment (pNCHS4 Rho–NLS–cre) containing human rhodopsin promoter was cloned. The Rho–Cre plasmid was digested with EcoR1 and I Ceu-1, and the 9.316 kb fragment containing the hRho promoter and Cre recombinase gel was purified. To generate transgenic mice, the purified DNA fragment was injected into fertilized oocytes according to standard protocols. ROSA26R reported the steady expression of Rho–Cre especially in photoreceptor cells, allowing further excising proteins in rod photoreceptors across the retina. This Rho–Cre transgenic line should thus prove useful as a general deletor line for genetic analysis of diverse aspects of retinopathy.     

Coordinate lentiviral expression of Cre recombinase and RFP/EGFP mediated by FMDV 2A and analysis of Cre activity

The site-specific recombination mediated by Cre recombinase has been utilized extensively in genetic engineering and gene function studies. Efficient delivery of a Cre enzyme with enzymatic activity and the ability to monitor the enzyme expression are required in applications, and lentiviral constructs with a fluorescent protein (FP) to report the Cre expression are suitable for most studies. However, the current lentiviral vector systems have some deficiencies in precise reporting the Cre expression through fluorescence. To solve the problem, we generated a lentiviral system with Cre and RFP or EGFP bridged by an FMDV 2A sequence in an open reading frame expressed by a CMV promoter. We then examined the capabilities of the constructs to package with VSVG into infectious virus and to mediate expression of the Cre enzyme and fluorescent reporter. Furthermore, we monitored the bioactivities of the expressed products. We demonstrated the coordinate expression of the enzyme and the reporter. The expressed Cre was efficient at removing LoxP-flanked fragments in cells and did not show obvious cellular toxicity, and the expressed FPs allowed direct observation under fluorescent microscope. Therefore, the conjugation of CMV-Cre-2A-FP represents a significant improvement to the current lentiviral Cre delivery systems for obtaining a required Cre activity while accurately monitoring its presence. Our study also provides information concerning application of the established vector system.     

Generation of a Tenascin-C-CreER2 Knockin Mouse Line for Conditional DNA Recombination in Renal Medullary Interstitial Cells

Renal medullary interstitial cells (RMIC) are specialized fibroblast-like cells that exert important functions in maintaining body fluid homeostasis and systemic blood pressure. Here, we generated a RMIC specific tenascin-C promoter driven inducible CreER2 knockin mouse line with an EGFP reporter. Similar as endogenous tenascin-C expression, the reporter EGFP expression in the tenascin-C-CreER2^+/− mice was observed in the inner medulla of the kidney, and co-localized with COX2 but not with AQP2 or AQP1, suggesting selective expression in RMICs. After recombination (tenascin-C-CreER2^+/−/ROSA26-lacZ^+/− mice + tamoxifen), β-gal activity was restricted to the cells in the inner medulla of the kidney, and didn''t co-localize with AQP2, consistent with selective Cre recombinase activity in RMICs. Cre activity was not obvious in other major organs or without tamoxifen treatment. This inducible RMIC specific Cre mouse line should therefore provide a novel tool to manipulate genes of interest in RMICs.     

Regulation of Cre recombinase by ligand-induced complementation of inactive fragments

Cre recombinase is extensively used to engineer the genome of experimental animals. However, its usefulness is still limited by the lack of an efficient temporal control over its activity. To overcome this, we have developed DiCre, a regulatable fragment complementation system for Cre. The enzyme was split into two moieties that were fused to FKBP12 (FK506-binding protein) and FRB (binding domain of the FKBP12–rapamycin-associated protein), respectively. These can be efficiently heterodimerized by rapamycin. Several variants, based on splitting Cre at different sites and using different linker peptides, were tested in an indicator cell line. The fusion proteins, taken separately, had no recombinase activity. Stable transformants, co-expressing complementing fragments based on splitting Cre between Asn59 and Asn60, displayed low background activity affecting 0.05–0.4% of the cells. Rapamycin induced a rapid recombination, reaching 100% by 48–72 h, with an EC₅₀ of 0.02 nM. Thus, ligand-induced dimerization can efficiently regulate Cre, and should be useful to achieve a tight temporal control of its activity, such as in the case of the creation of conditional knock-out animals.     

Neural expression of alpha-internexin promoter in vitro and in vivo

alpha-Internexin is a 66 kDa neuronal intermediate filament protein found most abundantly in the neurons of the nervous systems during early development. To characterize the function of mouse alpha-internexin promoter, we designed two different expression constructs driven by 0.7 kb or 1.3 kb of mouse alpha-internexin 5'-flanking sequences; one was the enhanced green fluorescent protein (EGFP) reporter for monitoring specific expression in vitro, and the other was the cre for studying the functional DNA recombinase in transgenic mice. After introducing DNA constructs into non-neuronal 3T3 fibroblasts and a neuronal Neuro2A cell line by lipofectamine transfection, we observed that the expression of EGFP with 1.3 kb mouse alpha-internexin promoter was in a neuron-dominant manner. To establish a tissue-specific pattern in the nervous system, we generated a transgenic mouse line expressing Cre DNA recombinase under the control of 1.3 kb alpha-Internexin promoter. The activity of the Cre recombinase at postnatal day 1 was examined by mating the cre transgenic mice to ROSA26 reporter (R26R) mice with knock-in Cre-mediated recombination. Analyses of postnatal day 1 (P1) newborns showed that beta-galactosidase activity was detected in the peripheral nervous system (PNS), such as cranial nerves innervating the tongue and the skin as well as spinal nerves to the body trunk. Furthermore, X-gal-labeled dorsal root ganglionic (DRG) neurons showed positive for alpha-Internexin in cell bodies but negative in their spinal nerves. The motor neurons in the spinal cord did not exhibit any beta-galactosidase activity. Therefore, the cre transgene driven by mouse alpha-internexin promoter, described here, provides a useful animal model to specifically manipulate genes in the developing nervous system.     

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.     

A modified RMCE-compatible Rosa26 locus for the expression of transgenes from exogenous promoters

Generation of gain-of-function transgenic mice by targeting the Rosa26 locus has been established as an alternative to classical transgenic mice produced by pronuclear microinjection. However, targeting transgenes to the endogenous Rosa26 promoter results in moderate ubiquitous expression and is not suitable for high expression levels. Therefore, we now generated a modified Rosa26 (modRosa26) locus that combines efficient targeted transgenesis using recombinase-mediated cassette exchange (RMCE) by Flipase (Flp-RMCE) or Cre recombinase (Cre-RMCE) with transgene expression from exogenous promoters. We silenced the endogenous Rosa26 promoter and characterized several ubiquitous (pCAG, EF1α and CMV) and tissue-specific (VeCad, αSMA) promoters in the modRosa26 locus in vivo. We demonstrate that the ubiquitous pCAG promoter in the modRosa26 locus now offers high transgene expression. While tissue-specific promoters were all active in their cognate tissues they additionally led to rare ectopic expression. To achieve high expression levels in a tissue-specific manner, we therefore combined Flp-RMCE for rapid ES cell targeting, the pCAG promoter for high transgene levels and Cre/LoxP conditional transgene activation using well-characterized Cre lines. Using this approach we generated a Cre/LoxP-inducible reporter mouse line with high EGFP expression levels that enables cell tracing in live cells. A second reporter line expressing luciferase permits efficient monitoring of Cre activity in live animals. Thus, targeting the modRosa26 locus by RMCE minimizes the effort required to target ES cells and generates a tool for the use exogenous promoters in combination with single-copy transgenes for predictable expression in mice.     

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。     

Selective expression of an aP2/Fatty Acid Binding Protein4-Cre transgene in non-adipogenic tissues during embryonic development

Mouse strains expressing the site-specific Cre recombinase facilitate conditional ablation or activation of genomic sequences when one or several exons of a gene of interest are flanked by loxP sites. Recently, several strains targeting Cre expression to adipocytes have been developed using promoter sequences from the aP2 (Fatty Acid Binding Protein 4, FABP4) gene for adipose tissue-specific gene expression studies. aP2/FABP4 is predominantly expressed in adipose tissue, and while this promoter provides adipocyte-restricted expression postnatally, its expression throughout embryonic development had not been previously characterized. In this report, we demonstrate that the aP2-Cre transgene is expressed and consistently localized within the embryo from mid-gestation stage 9.5 dpc. By 15.5 dpc, β-gal activity was detected primarily in the brown adipose tissue, trigeminal ganglia, dorsal root ganglia, cartilage primordia and vertebrae. Immunofluorescence staining for Cre recombinase and FABP4 protein showed a corresponding staining pattern similar to that of β-gal, confirming that Cre recombinase was produced in the transgenic line at late stages of development, and overlapped with endogenous aP2/FABP4 production. Further, fat-specific oil red O staining of tissue sections validated the presence of lipids in the stained tissues indicating that adipocytes and/or adipocyte-like cells were indeed present in these tissues. This is the first report to our knowledge to describe and confirm aP2/FABP4 promoter expression in this transgenic line during development in the mouse embryo and indicates that aP2/FABP4 expression occurs not only in mature adipocytes, but has a wider embryonic expression pattern than previously appreciated.Lucy Liaw and Deena Small contributed equally to this work     

Cell-specific expression of SERCA, the exogenous Ca2+ transport ATPase, in cardiac myocytes

We evaluated various constructs to obtain cell-specific expression of the sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) gene in cardiac myocytes after cDNA transfer by means of transfections or infections with adenovirus vectors. Expression of exogenous enhanced green fluorescent protein (EGFP) and SERCA genes was studied in cultured chicken embryo and neonatal rat cardiac myocytes, skeletal and smooth muscle cells, fibroblasts, and hepatocytes. Whereas the cytomegalovirus (CMV) promoter yielded high levels of protein expression in all cells studied, cardiac troponin T (cTnT) promoter segments demonstrated high specificity for cardiac myocytes. Their efficiency for protein expression was lower than that of the CMV promoter, but higher than that of cardiac myosin light chain or -myosin heavy chain promoter segments. A double virus system for Cre-dependent expression under control of the CMV promoter and Cre expression under control of a cardiac-specific promoter yielded high protein levels in cardiac myocytes, but only partial cell specificity due to significant Cre expression in hepatocytes. Specific intracellular targeting of gene products was demonstrated in situ by specific immunostaining of exogenous SERCA1 and endogenous SERCA2 and comparative fluorescence microscopy. The -374 cTnT promoter segment was the most advantageous of the promoters studied, producing cell-specific SERCA expression and a definite increase over endogenous Ca²⁺-ATPase activity as well as faster removal of cytosolic calcium after membrane excitation. We conclude that analysis of promoter efficiency and cell specificity is of definite advantage when cell-specific expression of exogenous SERCA is wanted in cardiac myocytes after cDNA delivery to mixed cell populations. cardiac myocytes; cell-specific expression; adenovirus vectors; calcium transport     

Tightly Regulated and Homogeneous Transgene Expression in Human Adipose-Derived Mesenchymal Stem Cells by Lentivirus with Tet-Off System

Genetic modification of human adipose tissue–derived multilineage progenitor cells (hADMPCs) is highly valuable for their exploitation in therapeutic applications. Here, we have developed a novel single tet-off lentiviral vector platform. This vector combines (1) a modified tetracycline (tet)-response element composite promoter, (2) a multi-cistronic strategy to express an improved version of the tet-controlled transactivator and the blasticidin resistance gene under the control of a ubiquitous promoter, and (3) acceptor sites for easy recombination cloning of the gene of interest. In the present study, we used the cytomegalovirus (CMV) or the elongation factor 1 α (EF-1α) promoter as the ubiquitous promoter, and EGFP was introduced as the gene of interest. hADMPCs transduced with a lentiviral vector carrying either the CMV promoter or the EF-1α promoter were effectively selected by blasticidin without affecting their stem cell properties, and EGFP expression was strictly regulated by doxycycline (Dox) treatment in these cells. However, the single tet-off lentiviral vector carrying the EF-1α promoter provided more homogenous expression of EGFP in hADMPCs. Intriguingly, differentiated cells from these Dox-responsive cell lines constitutively expressed EGFP only in the absence of Dox. This single tet-off lentiviral vector thus provides an important tool for applied research on hADMPCs.     

Production of a reporter transgenic pig for monitoring Cre recombinase activity

The pig is thought to be the most suitable non-human source of organs for xenotransplantation and is widely used as a model of human disease. Using pigs as disease models requires the design of conditional Cre recombinase-loxP gene modifications, which, in turn, requires a Cre-expressing pig with defined patterns of expression controlled by the use of a tissue-specific promoter. In order to monitor Cre recombinant expression in vivo, it is important to create a reporter strain. We have generated reporter a pig that is based on a single vector that drives the ubiquitous expression of the enhanced green fluorescent protein (EGFP). The EGFP gene is expressed only after Cre-mediated excision of loxP-flanked stop sequences. These reporter transgenic pigs will be of great value for monitoring Cre recombinase activity in vivo.     

Split‐Cre recombinase effectively monitors protein–protein interactions in living bacteria

The ability of Cre recombinase to excise genetic material has been used extensively for genome engineering in prokaryotic and eukaryotic cells. Recently, split‐Cre fragments have been described that advance control of recombinase activity in mammalian cells. However, whether these fragments can be utilized for monitoring protein‐protein interactions has not been reported. In this work, we developed a protein‐fragment complementation assay (PCA) based on split‐Cre for monitoring and engineering pairwise protein interactions in living Escherichia coli cells. This required creation of a dual‐fluorescent reporter plasmid that permits visualization of reconstituted Cre recombinase activity by switching from red to green in the presence of an interacting protein pair. The resulting split‐Cre PCA faithfully links cell fluorescence with differences in binding affinity, thereby allowing the facile isolation of high‐affinity binders based on phenotype. Given the resolution of its activity and sensitivity to interactions, our system may prove a viable option for poorly expressed or weakly interacting protein pairs that evade detection in other PCA formats. Based on these findings, we anticipate that our split‐Cre PCA will become a highly complementary and useful new addition to the protein‐protein interaction toolbox.     

Postnatal male germ‐cell expression of cre recombinase in Tex101‐iCre transgenic mice

We have generated a transgenic mouse line that expresses improved Cre recombinase (iCre) under the control of the testis‐expressed gene 101 (Tex101) promoter. This transgenic mouse line was named Tex101‐iCre. Using the floxed ROSA reporter mice, we found that robust Cre recombinase activity was detected in postnatal testes with weak or no activity in other tissues. Within the testis, Cre recombinase was active in spermatogenic cells as early as the prospermatogonia stage at day 1 after birth. In 30‐ and 60‐day‐old mice, positive Cre recombinase activity was detected not only in prospermatogonia but also in spermatogenic cells at later stages of spermatogenesis. There was little or no Cre activity in interstitial cells. Breeding wild‐type females with homozygous floxed fibroblast growth factor receptor 2 (Fgfr2) males carrying the Tex101‐iCre transgene did not produce any progeny with the floxed Fgfr2 allele. All the progeny inherited a recombined Fgfr2 allele, indicating that complete deletion of the floxed Fgfr2 allele by Tex101‐iCre can be achieved in the male germline. Furthermore, FGFR2 protein was not detected in spermatocytes and spermatids of adult Fgfr2^fl/fl;Tex101‐iCre mice. Taken together, our results suggest that the Tex101‐iCre mouse line allows the inactivation of a floxed gene in spermatogenic cells in adult mice, which will facilitate the functional characterization of genes in normal spermatogenesis and male fertility. genesis 48:717–722, 2010. © 2010 Wiley‐Liss, Inc.     

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.