Cre stoplight: a red/green fluorescent reporter of Cre recombinase expression in living cells.

The Cre/lox system is a powerful genetic tool with which to manipulate the genome. Here, we describe the development of a simple reporter system for Cre recombinase, called the Cre Stoplight. In the absence of Cre, the red fluorescent protein is expressed; when Cre catalyzes a recombination event, the green fluorescent protein is produced. Testing this system in transiently transfected cells showed that it produced robust signals (90% of the cells converted from red to green) when equal amounts of the plasmids encoding Cre recombinase and the Cre Stoplight were used. A 1:100 ratio of enzyme to reporter plasmid produced similar results, and a 1:10000 ratio was necessary to significantly reduce the number of cells converting to green (1%).     

Expression of reg/PSP, a pancreatic exocrine gene: relationship to changes in islet beta-cell mass

A cDNA termed reg was recently isolated by differential screening of a library prepared from regenerating islets isolated from pancreatic remnants of rats subjected to 90% pancreatectomy and nicotinamide treatment. This led to speculation that this gene may be involved in expansion of beta-cell mass. In the current study we have measured reg expression after implantation and resection of a solid insulinoma tumor into rats, maneuvers known, respectively, to reduce and reexpand the volume of beta-cells in the islet. Animals with an implanted insulinoma tumor became profoundly hypoglycemic. Islet beta-cells declined from the normal 75% of total islet volume to less than 30%, in concert with a marked reduction in the reg mRNA level. Removal of the tumor resulted in a sharp increase in beta-cell replication, as measured by [3H]thymidine incorporation and a return to normal beta-cell volume within 4 days of tumor resection. This was associated with a transient induction in reg expression compared to that in tumor-bearing animals, effectively returning the amount of reg mRNA to the levels found in normal animals within 48 h; at later time points after tumor removal (3-7 days) reg expression declined, but then rose toward normal. In situ hybridization analysis localized the initial induction in reg mRNA expression to the exocrine pancreas. Continuous infusion of insulin into normal rats for 4 days, a maneuver that does not significantly reduce beta-cell mass, resulted in dramatically reduced insulin mRNA in islets, but no change in the levels of reg mRNA. We conclude that the diminution in pancreatic beta-cell mass caused by subcutaneous implantation of an insulinoma is associated with reduced reg gene expression and that the increase in beta-cell replication after resection of the tumor is preceded by return of reg gene expression toward normal.     

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.     

Oligodendroglial and pan‐neural crest expression of Cre recombinase directed by Sox10 enhancer

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.     

VAChT-Cre. Fast and VAChT-Cre.Slow: postnatal expression of Cre recombinase in somatomotor neurons with different onset

The cholinergic gene locus (CGL) consists of the genes encoding the choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter (VAChT). To establish a cholinergic-specific Cre-expressing mouse, we constructed a transgene expression vector (VAChT-Cre) with 11.3 kb human CGL in which a Cre-IRES-EGFP unit was inserted in the VAChT open reading frame. The activity of Cre, whose expression was driven by the VAChT promoter, was examined by crossing a reporter mouse (CAG-CAT-Z) in which expression of LacZ is activated upon Cre-mediated recombination. Transgenic lines with the VAChT-Cre construct displayed the restricted Cre expression in a subset of cholinergic neurons in the somatomotor nuclei and medial habenular nucleus, but absent in visceromotor and other central and peripheral cholinergic neurons. Cre expression was first observed at postnatal day 7 and later detected in approximately 40-60% of somatomotor neurons. Based on the onset of Cre expression, we generated two mouse lines (two alleles; VAChT-Cre. Fast and VAChT-Cre.Slow) in which Cre expression reaches maximal levels fast and slow, respectively. The use of VAChT-Cre mice should allow us to deliver Cre to a subset of postnatal motor neurons, thereby bypassing lethality and facilitating analysis of gene function in adult motor neurons.     

Differential oocyte-specific expression of Cre recombinase activity in GDF-9-iCre, Zp3cre, and Msx2Cre transgenic mice

Oocyte-specific deletion of ovarian genes using Cre/loxP technology provides an excellent tool to understand their physiological roles during folliculogenesis, oogenesis, and preimplantation embryonic development. We have generated a transgenic mouse line expressing improved Cre recombinase (iCre) driven by the mouse growth differentiation factor-9 (GDF-9) promoter. The resulting transgenic mouse line was named GDF-9-iCre mice. Using the floxed ROSA reporter mice, we found that Cre recombinase was expressed in postnatal ovaries, but not in heart, liver, spleen, kidney, and brain. Within the ovary, the Cre recombinase was exclusively expressed in the oocytes of primordial follicles and follicles at later developmental stages. The expression of iCre of GDF-9-iCre mice was shown to be earlier than the Cre expression of Zp3Cre and Msx2Cre mice, in which the Cre gene is driven by zona pellucida protein 3 (Zp3) promoter and a homeobox gene Msx2 promoter, respectively, in the postnatal ovary. Breeding wild-type males with heterozygous floxed germ cell nuclear factor (GCNF) females carrying the GDF-9-iCre transgene did not produce any progeny having the floxed GCNF allele, indicating that complete deletion of the floxed GCNF allele can be achieved in the female germline by GDF-9-iCre mice. These results suggest that GDF-9-iCre mouse line provides an excellent genetic tool for understanding functions of oocyte-expressing genes involved in folliculogenesis, oogenesis, and early embryonic development. Comparison of the ontogeny of the Cre activities of GDF-9-iCre, Zp3Cre, and Msx2Cre transgenic mice shows there is sequential Cre activity of the three transgenes that will allow inactivation of a target gene at different points in folliculogenesis.     

Biphasic response of pancreatic beta-cell mass to ablation of tuberous sclerosis complex 2 in mice

Recent studies have demonstrated the importance of insulin or insulin-like growth factor 1 (IGF-1) for regulation of pancreatic beta-cell mass. Given the role of tuberous sclerosis complex 2 (TSC2) as an upstream molecule of mTOR (mammalian target of rapamycin), we examined the effect of TSC2 deficiency on beta-cell function. Here, we show that mice deficient in TSC2, specifically in pancreatic beta cells (betaTSC2(-/-) mice), manifest increased IGF-1-dependent phosphorylation of p70 S6 kinase and 4E-BP1 in islets as well as an initial increased islet mass attributable in large part to increases in the sizes of individual beta cells. These mice also exhibit hypoglycemia and hyperinsulinemia at young ages (4 to 28 weeks). After 40 weeks of age, however, the betaTSC2(-/-) mice develop progressive hyperglycemia and hypoinsulinemia accompanied by a reduction in islet mass due predominantly to a decrease in the number of beta cells. These results thus indicate that TSC2 regulates pancreatic beta-cell mass in a biphasic manner.     

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.     

Quasi-equivalence in site-specific recombinase structure and function: crystal structure and activity of trimeric Cre recombinase bound to a three-way Lox DNA junction

The crystal structure of a novel Cre-Lox synapse was solved using phases from multiple isomorphous replacement and anomalous scattering, and refined to 2.05 A resolution. In this complex, a symmetric protein trimer is bound to a Y-shaped three-way DNA junction, a marked departure from the pseudo-4-fold symmetrical tetramer associated with Cre-mediated LoxP recombination. The three-way DNA junction was accommodated by a simple kink without significant distortion of the adjoining DNA duplexes. Although the mean angle between DNA arms in the Y and X structures was similar, adjacent Cre trimer subunits rotated 29 degrees relative to those in the tetramers. This rotation was accommodated at the protein-protein and DNA-DNA interfaces by interactions that are "quasi-equivalent" to those in the tetramer, analogous to packing differences of chemically identical viral subunits at non-equivalent positions in icosahedral capsids. This structural quasi-equivalence extends to function as Cre can bind to, cleave and perform strand transfer with a three-way Lox substrate. The structure explains the dual recognition of three and four-way junctions by site-specific recombinases as being due to shared structural features between the differently branched substrates and plasticity of the protein-protein interfaces. To our knowledge, this is the first direct demonstration of quasi-equivalence in both the assembly and function of an oligomeric enzyme.     

Circadian and age-dependent expression patterns of GLUT2 and glucokinase in the pancreatic beta-cell of diabetic and nondiabetic rats.

Alterations in glucose sensing are well-known in both humans and animal models of non-insulin-dependent diabetes mellitus. However, the circadian- and age-dependent expression of glucose-sensing genes has not previously been investigated in vivo. In the present paper, we show a progressive loss of beta-cell GLUT2-mRNA and, by immunocytochemistry, a gain of soluble, cytoplasmic GLUT2-protein in Goto-Kakizaki rat islets. We report that GLUT2-mRNA shows significant diurnal variation, which is stronger in metabolically healthy rats. We also demonstrate the significant diurnal variation of glucokinase-mRNA, with higher levels in the pancreas of 6-week-old Goto-Kakizaki rats than in Wistar rats. This leads to a maximum glucose phosphorylation capacity in-phase with food intake, enhanced glucose-stimulated insulin secretion, and prevents postprandial hyperglycemia. Perfusion experiments showed a reduction in glucose-stimulated insulin secretion in Goto-Kakizaki rat islets with an impaired first phase. Hyperglycemia and hypoinsulinemia in newborn and up to 3-week-old Goto-Kakizaki rats are thus probably due to reduced pancreatic beta-cell content, reduced beta-cell insulin content and impaired glucose sensing. The de-compensation of the metabolic situation in 42-week-old Goto-Kakizaki rats is likely to be caused by beta-cell destruction accompanied by negligible accumulation of GLUT2 in the cell membrane and further reduction of glucokinase expression.     

Site-specific recombination mediated by an adenovirus vector expressing the Cre recombinase protein: a molecular switch for control of gene expression.

We have constructed replication-defective human adenovirus (Ad) type 5 vectors containing the gene for the Cre recombinase from bacteriophage P1 under control of the human cytomegalovirus immediate-early promoter (AdCre). Expression of the protein was detected in replication-permissive (293) and in nonpermissive (MRC5) cell lines, and its biochemical activity was demonstrated in a cell-free recombination assay using a plasmid containing two loxP sites. To study Cre-mediated recombination in an intracellular system, we constructed an Ad vector (AdMA19) containing the luciferase cDNA under control of the human cytomegalovirus promoter but separated from it by an extraneous spacer sequence flanked by loxP sites which blocked luciferase expression. Upon coinfection of 293 or MRC5 cells with AdMA19 and AdCre, luciferase expression was specifically induced by Cre-mediated excision of the intervening sequence. The use of Ad vectors combined with the Cre-loxP system for regulation of gene expression and other possible applications is discussed.     

Single islet beta-cell stimulation by nutrients: relationship between pyridine nucleotides, cytosolic Ca2+ and secretion.

It is generally believed that the initiation of insulin secretion by nutrient stimuli necessitates the generation of metabolic coupling factors, leading to membrane depolarization and the gating of voltage-sensitive Ca2+ channels. To establish this sequence of events, the kinetics of endogenous fluorescence of reduced pyridine nucleotides [NAD(P)H], reflecting nutrient metabolism, were compared to those of cytosolic calcium ([Ca2+]i) rises in single cultured rat islet beta-cells. In preliminary experiments, the loss of quinacrine fluorescence from prelabelled cells was used as an indicator of secretion. This dye is concentrated in the acidic insulin-containing secretory granules. Both glucose and 2-ketoisocaproate (KIC) raised [Ca2+]i in a dose-dependent manner. There was marked cellular heterogeneity in the [Ca2+]i response patterns. The two nutrient stimuli also increased NAD(P)H fluorescence, again showing cell-to-cell variations. In combined experiments, where the two parameters were measured in the same cell, the elevation of the NAD(P)H fluorescence preceded the rise in [Ca2+]i, confirming the statistical evaluation performed on separate cells. The application of two consecutive glucose challenges revealed coordinated changes in [Ca2+]i and NAD(P)H fluorescence. Finally, quinacrine secretion was stimulated by two nutrients with onset times similar to those recorded for [Ca2+]i elevations. These results clearly demonstrate that increased metabolism occurs during the lag period preceding Ca2+ influx via voltage-sensitive Ca2+ channels, a prerequisite for the triggering of insulin secretion by nutrient stimuli.     

Alpha-adrenergic inhibition of rat pancreatic beta-cell replication and insulin secretion is mediated through a pertussis toxin-sensitive G-protein regulating islet cAMP content.

The rate of DNA synthesis, insulin secretion and cAMP content in isolated pancreatic islets were markedly inhibited by long-term exposure to the alpha 1-adrenoceptor agonist phenylephrine, the alpha 2-adrenoceptor agonist clonidine and the beta-adrenoceptor antagonist propranolol. Pertussis toxin or the stimulatory cAMP analog Sp-cAMPS increased DNA synthesis and insulin secretion in the absence of the adrenergic agents. Pertussis toxin blocked the inhibitory actions of these agents on DNA synthesis, insulin secretion and cAMP content, and a similar protection was imposed by Sp-cAMPS. Thus, long-term alpha-adrenergic stimulation interferes with signaling through pertussis toxin-sensitive G-protein(s) and, by decreasing the islet cAMP content, inhibits beta-cell DNA synthesis and insulin secretion.     

Selective deletion of Pten in pancreatic beta cells leads to increased islet mass and resistance to STZ-induced diabetes

Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a lipid phosphatase. PTEN inhibits the action of phosphatidylinositol-3-kinase and reduces the levels of phosphatidylinositol triphosphate, a crucial second messenger for cell proliferation and survival, as well as insulin signaling. In this study, we deleted Pten specifically in the insulin producing beta cells during murine pancreatic development. Pten deletion leads to increased cell proliferation and decreased cell death, without significant alteration of beta-cell differentiation. Consequently, the mutant pancreas generates more and larger islets, with a significant increase in total beta-cell mass. PTEN loss also protects animals from developing streptozotocin-induced diabetes. Our data demonstrate that PTEN loss in beta cells is not tumorigenic but beneficial. This suggests that modulating the PTEN-controlled signaling pathway is a potential approach for beta-cell protection and regeneration therapies.