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Results for “cellular engineering”

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PubMed2003

Eradication of pathogenic beta-catenin by Skp1/Cullin/F box ubiquitination machinery.

The use of Skp1/Cull 1/F box (SCF) ubiquitin-conjugation machinery as a potential knockout tool offers a means of eradicating disease-causing proteins. Here a chimeric F box protein (CFP) was engineered to achieve selective eradication of pathogenic beta-catenin in colorectal cancer. We show that CFP specifically searches for and subsequently links the abnormal beta-catenin to the cellular SCF ubiquitination complex. Introduction of the CFP to colorectal cancer cells induced targeted ubiquitination and proteolytic degradation of nuclear and cytoplasmic free beta-catenin while preserving its normal cellular adhesion counterpart. Elimination of pathogenic beta-catenin suppressed constitutive Wingless/Wnt signaling and inhibited in vitro and in vivo tumor cell growth. This study demonstrates a practical utility of a SCF-based knockout system as a tool in targeting an abnormal protein that affects growth and transformation.

Cell Division↗

PubMed2006

Evaluation of engineered meniscal cartilage constructs based on different scaffold geometries using magnetic resonance imaging and spectroscopy.

Magnetic resonance imaging (MRI) and spectroscopy (MRS) were used to evaluate the properties different scaffold geometries for the production of bioartificial meniscal cartilage constructs. Engineered were generated in perfusion bioreactors from mature sheep meniscal fibrochondrocytes,scaffolds cut from a knitted polyethylene therephtalate (PET) fabric, with a distribution of fibers, 50 microm pores and a density of 45 mg/cm(3) (NF scaffolds), and from two versions of this fabric, which included larger pore sizes (1500 x 500 microm(2)) and densities of (sIV scaffolds) and 83 mg/cm(3) (sV scaffolds). MRI methods were used to determine the permeability the constructs to a low molecular weight MR contrast agent and to measure the macroscopic of medium through and around the constructs. These parameters were correlated with measurements of cell growth and cellular energetics. Cell-free sIV scaffolds were 2- and 5-more porous to flow than the empty sV and NF scaffolds, respectively. These scaffolds, after days of cell growth, were also more permeable to an MR contrast agent. sIV scaffolds yielded(n = 9) with higher cellularities (41 +/- 1%) compared with NF (32 +/- 1%, p < 0.0001) and sV (30 +/- 1%, p < 0.0001) and, when normalized to cell numbers, demonstrated proportionally levels of nucleoside triphosphates (NTP), indicating increased cell viability. Scaffold geometry a marked effect on the properties of engineered meniscal cartilage. MRI and MRS are powerful techniques that can be used to optimize the design of engineered meniscal cartilage and that could be used subsequently to evaluate clinical outcome postimplantation.

Animals↗

PubMed2004

Genetic analysis of the neuronal and ubiquitous AP-3 adaptor complexes reveals divergent functions in brain.

Neurons express adaptor (AP)-3 complexes assembled with either ubiquitous (beta3A) or neuronal-specific (beta3B) beta3 isoforms. However, it is unknown whether these complexes indeed perform distinct functions in neuronal tissue. Here, we explore this hypothesis by using genetically engineered mouse models lacking either beta3A- or beta3B-containing AP-3 complexes. Somatic and neurological phenotypes were specifically associated with the ubiquitous and neuronal adaptor deficiencies, respectively. At the cellular level, AP-3 isoforms were localized to distinct neuronal domains. beta3B-containing AP-3 complexes were preferentially targeted to neuronal processes. Consistently, beta3B deficiency compromised synaptic zinc stores assessed by Timm's staining and the synaptic vesicle targeting of membrane proteins involved in zinc uptake (ZnT3 and ClC-3). Surprisingly, despite the lack of neurological symptoms, beta3A-deficient mouse brain possessed significantly increased synaptic zinc stores and synaptic vesicle content of ZnT3 and ClC-3. These observations indicate that the functions of beta3A- and beta3B-containing complexes are distinct and divergent. Our results suggest that concerted nonredundant functions of neuronal and ubiquitous AP-3 provide a mechanism to control the levels of selected membrane proteins in synaptic vesicles.

Adaptor Protein Complex 3↗

PubMed2003

Arterial wall adaptation under elevated longitudinal stretch in organ culture.

Arteries in vivo are subjected to large longitudinal stretch which may change significantly due to vascular disease and surgery. However, little is known about the effect of longitudinal stretch on vascular function and wall remodeling, although the effects of tensile and shear stress from blood pressure and flow have been well documented. To study the effect of longitudinal stretch on vascular function and wall remodeling, porcine carotid arteries were longitudinally stretched 20% more than in vivo for 5 days while being maintained in an ex vivo organ culture system under conditions of pulsatile flow at physiologic pressure. Vessel viability was demonstrated by strong vasomotor responses to norepinephrine (NE, 10(-6) M), carbachol (10(-6) M), and sodium nitroprusside (10(-5) M), as well as by dense staining for mitochondrial activity and a low occurrence of cell necrosis. Cell proliferation was examined by incorporation of bromodeoxyuridine (BrdU). Results showed that arteries maintain normal structure and viability after 5 days in organ culture. Both the stretched and control arteries demonstrated significant contractile responses. For example, both stretched and control arteries showed approximately 10% diameter contraction in response to NE. Stretched arteries contained 8% BrdU-positive cells compared to 5% in controls (p<0.05). These results indicate that longitudinal stretch promotes cell proliferation in arteries while maintaining arterial function.

Adaptation, Physiological↗

PubMed2001

Preexisting immunity to poliovirus does not impair the efficacy of recombinant poliovirus vaccine vectors.

Recombinant viruses are attractive candidates for the development of novel vaccines. A number of viruses have been engineered as vaccine vectors to express antigens from other pathogens or tumors. Inoculation of susceptible animals with this type of recombinant virus results in the induction of both humoral and cellular immune responses directed against the foreign antigens. A general problem to this approach is that existing immunity to the vector can diminish or completely abolish the efficacy of the viral vector. In this study, we investigated whether poliovirus recombinants are capable of inducing effective immunity to the foreign antigen in previously vaccinated animals. Antipoliovirus immunity was induced in susceptible mice by intraperitoneal immunization with live poliovirus. Immunized mice developed antibodies directed against capsid proteins that effectively neutralized poliovirus in vitro and protected animals from a lethal challenge with a high dose of pathogenic poliovirus. To test whether preexisting immunity reduces the efficacy of vaccination with recombinant poliovirus, immunized mice were inoculated with a recombinant poliovirus expressing the C-terminal half of chicken ovalbumin (Polio-Ova). Animals developed ovalbumin-specific antibodies and cytotoxic T lymphocytes (CTL). While the antibody titers observed in preimmune and naive mice were similar, the overall CTL response appeared to be reduced in preimmune mice. Importantly, vaccination with Polio-Ova was able to effectively protect preimmune mice against lethal challenge with a tumor expressing the antigen. Thus, preexisting immunity to poliovirus does not compromise seriously the efficacy of replication-competent poliovirus vaccine vectors. These results contrast with those observed for other viral vaccine vectors and suggest that preexisting immunity does not equally affect the vaccine potential of individual viral vectors.

Animals↗

PubMed2004

Herpesvirus/retrovirus chimeric vectors.

Over the years, the design of HSV-1 based vectors has developed from different types of replicative-defective and replication-conditioned recombinant viruses to plasmid based amplicon vectors. More recently hybrid or chimeric vectors have incorporated desired elements of different viruses to increase the efficacy of gene delivery in vitro and in vivo. Amongst different systems, herpesvirus/retrovirus chimeras take advantage of the features of the HSV-1 vectors to efficiently transduce large amounts of foreign genetic sequences, remaining episomal, to allow production of recombinant retrovirus vectors able to stably integrate into the cellular genome. This review will focus on three different groups of herpesvirus/retrovirus chimeric vectors aimed to: generate retrovirus particles in cells tranduced with HSV-1 amplicon vectors; express a limited set of retrovirus genes for vaccine purposes; and express herpesvirus/retrovirus chimeric proteins to study cellular targeting signal and improve their biological effect.

Animals↗

PubMed2000

[Extension of life-span of normal human fibroblasts by reconstitution of telomerase activity].

Most of normal human somatic cells can divide only a finite number of times and inevitably become senescent. Telomerase is an enzyme that imparts replicative immortality by maintaining the length of the telomeres when expressed in reproductive and cancer cells. Cells that are mortal do not express the telomerase. Recently it was reported that the life-span of the normal human cells could be successfully extended by introduction of telomerase into these cells. We have found, in the previous work, that fibroblasts exhibited an osteogentic potential, and therefore, can be considered as a type of "seed cells" in tissue engineering for bone repairing and reconstruction. But this potential was impaired by the limitation in life-span and proliferative capacity of the normal fibroblasts. In the present work, plasmid pGRN145 bearing a cDNA insert of human telomerase reverse transcriptase (hTERT) was introduced into the fibroblasts with osteogenic potential by electroporation. The stable hTERT+ fibroblast clones was established and cultured for long-term in a medium containing hygromycin-B. The exogenous hTERT mRNA expression and telomerase activity were detected. The hTERT+ fibroblasts showed shorter population doubling time and no beta-galactosidase stain, which indicated a stronger proliferative capacity and fewer signs of cell senescence, compared to their hTERT- counterpart. These evidenced that the life-span of hTERT+ fibroblasts was extended. The assays for DNA euploidy by flow cytometry and chromosome karyotype by cytogenetic technique showed no signs of heteroploidy, providing the data for cell carcinogenesis and utilization safety. The results of the present study suggested that the introduction of hTERT could make the life-span of normal fibroblast extended without causing their malignant transformation, and such type of "longevous" fibroblasts might be clinically useful in tissue engineering for bone repairing and reconstruction.

Cells, Cultured↗

PubMed1996

Differentiation induced by the c-Mpl cytokine receptor is blocked by mutant Shc adaptor protein.

c-Mpl, a member of the cytokine receptor superfamily, induces both proliferative and differentiation responses when stimulated with its ligand thrombopoietin (TPO). To examine signal transduction pathways associated with differentiation versus proliferation, 32D clone 3 cells, a murine interleukin 3-(IL-3)-dependent cell line capable of granulocytic differentiation, were engineered to express human c-Mpl (designated 32DM.2). Human TPO-containing medium was produced by transient transfection of 293 cells. Treatment of 32DM.2 cells with human TPO induced cellular aggregates within 12 h of exposure to ligand. 32DM.2 cells maintained in the presence of TPO did not change in cell number over a 72-h period and acquired characteristics of granulocytic differentiation as evidenced by metamyelocytic cellular morphology. The differentiation effect of TPO was observed in the absence and presence of the mitogen IL-3. Evaluation of protein tyrosine phosphorylation following exposure to ligand revealed that TPO stimulation induced an elevated level of tyrosine phosphorylation of the adaptor protein Shc when compared with IL-3. However, treatment of 32DM.2 cells with TPO did not result in the phosphorylation of mitogen-activated protein kinase (MAPK). To evaluate the potential role of Shc in c-Mpl differentiation, we transfected 32DM.2 cells with a mutant Shc gene that lacked the region coding for the phosphotyrosine interaction domain (delta PI-Shc). Expression of the delta PI-Shc protein in 32DM.2 cells blocked the TPO differentiation response with no effect on IL-3-stimulated proliferation. These studies demonstrate that c-Mpl-induced differentiation results from the activation of signal transduction pathways that are dominant to the IL-3 proliferative response and independent of the Ras/MAPK signal transduction pathway. The ability of the delta PI-Shc protein to block TPO-induced differentiation implicates Shc as a mediator of signal transduction pathways leading to differentiation, which is distinct from its role as a mediator in activating the Ras/MAPK pathway.

Animals↗

PubMed2006

Characterization of a novel polymeric scaffold for potential application in tendon/ligament tissue engineering.

Unlike braided fabrics, knitted scaffolds have been proven to favor deposition of collagenous connective tissue matrix, which is crucial for tendon/ligament reconstruction. But cell seeding of such scaffolds often requires a gel system, which is unstable in a dynamic situation, especially in the knee joint. This study developed a novel, biodegradable nano-microfibrous polymer scaffold by electrospinning PLGA nanofibers onto a knitted PLGA scaffold in order to provide a large biomimetic surface for cell attachment. Porcine bone marrow stromal cells were seeded onto either the novel scaffolds by pipetting a cell suspension (Group I) or the knitted PLGA scaffolds by immobilizing in fibrin gel (Group II). Cell attachment at 36 hours, cell proliferation and extracellular matrix synthesis at 1 week, and mechanical properties over 2 weeks were investigated. Cell attachment was comparable and cell proliferation was faster in Group I. Moreover, cellular function was more actively exhibited in Group I, as evident by the higher expression of collagen I, decorin, and biglycan genes. Thus, this novel scaffold, facilitating cell seeding and promoting cell proliferation, function, and differentiation, could be applied with promise in tissue engineering of tendon/ligament.

Animals↗

PubMed2005

Novel tissue-engineered biodegradable material for reconstruction of vascular wall.

BACKGROUND: To solve several problems with artificial grafts, we sought to develop a novel bioengineered material that can promote tissue regeneration without ex vivo cell seeding and that has sufficient durability to be used for artery reconstruction. Here, we tested whether this biodegradable material could accelerate the in situ regeneration of autologous cardiovascular tissue, especially of the arterial wall, in various models of cardiovascular surgeries. METHODS: The tissue-engineered patch was fabricated by compounding a collagen-microsponge with a biodegradable polymeric scaffold composed of polyglycolic acid knitted mesh, reinforced on the outside with woven polylactic acid. Tissue-engineered patches without precellularization were grafted into the porcine descending aorta (n = 5), the porcine pulmonary arterial trunk (n = 8), or the canine right ventricular outflow tract (as the large graft model; n = 4). Histologic and biochemical assessments were performed 1, 2, and 6 months after the implantation. RESULTS: There was no thrombus formation in any animal. Two months after grafting, all the grafts showed good in situ cellularization by hematoxylin/eosin and immunostaining. The quantification of the cell population by polymerase chain reaction showed a large number of endothelial and smooth muscle cells 2 months after implantation. In the large graft model, the architecture of the patch was similar to that of native tissue 6 months after implantation. CONCLUSIONS: A tissue-engineered patch made of our biodegradable polymer and collagen-microsponge provided good in situ regeneration at both the venous and arterial wall, suggesting that this patch can be used as a novel surgical material for the repair of the cardiovascular system.

Absorbable Implants↗

PubMed1997

Glutathione reductase turned into trypanothione reductase: structural analysis of an engineered change in substrate specificity.

Trypanosoma and Leishmania, pathogens responsible for diseases such as African sleeping sickness, Chagas' heart disease, or Oriental sore, are two of the very few genera that do not use the ubiquitous glutathione/glutathione reductase system to keep a stable cellular redox balance. Instead, they rely on trypanothione and trypanothione reductase to protect them from oxidative stress. Trypanothione reductase (TR) and the corresponding host enzyme, human red blood cell glutathione reductase (GR), belong to the same flavoprotein family. Despite their closely related three-dimensional structures and although their natural substrates share the common structural glutathione core, the two enzymes are mutually exclusive with respect to their disulfide substrates. This makes the parasite enzyme a potential target for antitrypanosomal drug design. While a large body of structural data on GR complexes is available, information on TR-ligand interactions is very limited. When the two amino acid changes Ala34Glu and Arg37Trp are introduced into human GR, the resulting mutant enzyme (GRTR) prefers trypanothione 700-fold over its original substrate, effectively converting a GR into a TR [Bradley, M., Bücheler, U. S., & Walsh, C. T. (1991) Biochemistry 30, 6124-6127]. The crystal structure of GRTR has been determined at 2.3 A resolution and refined to a crystallographic R factor of 20.9%. We have taken advantage of the ease with which ligand complexes can be produced in GR crystals, a property that extends to the isomorphous GRTR crystals, and have produced and analyzed crystals of GRTR complexes with glutathione, trypanothione, glutathionylspermidine and of a true catalytic intermediate, the mixed disulfide between trypanothione and the enzyme. The corresponding molecular structures have been characterized at resolutions between 2.3 and 2.8 A with R factors ranging from 17.1 to 19.7%. The results indicate that the Ala34Glu mutation causes steric hindrance leading to a large displacement of the side chain of Arg347. This movement combined with the change in charge introduced by the mutations modifies the binding cavity, forcing glutathione to adopt a nonproductive binding mode and permitting trypanothione and to a certain degree also the weak substrate glutathionylspermidine to assume a productive mode.

Animals↗

PubMed2004

Tissue-engineered microvessels on three-dimensional biodegradable scaffolds using human endothelial progenitor cells.

Tissue engineering may offer patients new options when replacement or repair of an organ is needed. However, most tissues will require a microvascular network to supply oxygen and nutrients. One strategy for creating a microvascular network would be promotion of vasculogenesis in situ by seeding vascular progenitor cells within the biopolymeric construct. To pursue this strategy, we isolated CD34(+)/CD133(+) endothelial progenitor cells (EPC) from human umbilical cord blood and expanded the cells ex vivo as EPC-derived endothelial cells (EC). The EPC lost expression of the stem cell marker CD133 but continued to express the endothelial markers KDR/VEGF-R2, VE-cadherin, CD31, von Willebrand factor, and E-selectin. The cells were also shown to mediate calcium-dependent adhesion of HL-60 cells, a human promyelocytic leukemia cell line, providing evidence for a proinflammatory endothelial phenotype. The EPC-derived EC maintained this endothelial phenotype when expanded in roller bottles and subsequently seeded on polyglycolic acid-poly-l-lactic acid (PGA-PLLA) scaffolds, but microvessel formation was not observed. In contrast, EPC-derived EC seeded with human smooth muscle cells formed capillary-like structures throughout the scaffold (76.5 +/- 35 microvessels/mm(2)). These results indicate that 1) EPC-derived EC can be expanded in vitro and seeded on biodegradable scaffolds with preservation of endothelial phenotype and 2) EPC-derived EC seeded with human smooth muscle cells form microvessels on porous PGA-PLLA scaffolds. These properties indicate that EPC may be well suited for creating microvascular networks within tissue-engineered constructs.

AC133 Antigen↗

PubMed1998

Construction of engineering host by non-random method in Saccharomyces cerevisiae.

A yeast engineering host, GJ30, was constructed by a non-random method. GJ30 derivated from wild type yeast have leu2- and ura3- deficients which can be used as selection markers. The PCR analysis showed that the homologous recombinations occurred in the leu2 and ura3 locus of GJ30, respectively. The biological properties (cellular growth density, stability and efficiency for gene expression) of GJ30 were compared with other several yeast stains. The results showed that GJ30 is a suitable host strain for expression of foreign genes.

3-Isopropylmalate Dehydrogenase↗

PubMed2006

Analyzing protein kinase dynamics in living cells with FRET reporters.

Genetically encoded reporters based on fluorescence resonance energy transfer (FRET) are being developed for analyzing spatiotemporal dynamics of kinase activities in living cells, as the activities of this class of enzymes are often dynamically regulated and spatially compartmentalized within specific signaling context. Here we describe a general modular design and engineering strategies for the development of activity reporters for kinases of interest, using A-kinase activity reporter (AKAR) as an illustrative example. Discussed here are basic structure of such reporters, design considerations, reporter gene construction, cellular and in vitro characterization. Strategies for improving specificity, dynamic range or sensitivity, reversibility and integrity of the reporter as well as basic methods for live-cell time-lapse imaging using these reporters are summarized. Discussion of using this approach in the study of MAPK cascades is also provided. These FRET-based kinase activity reporters, along with analogous probes based on alternative designs, provide real-time tracking of kinase dynamics with subcellular resolution, which should complement other methods and offer great opportunities to delineate the molecular mechanisms underlying the complex regulation of kinases.

Animals↗

PubMedDate not supplied

Human chondrocytes and mesenchymal stem cells grown onto engineered scaffold.

The development of improved methods for treatment of chondral defects using autologous cells in combination with biomaterials leads to a new generation of implantable devices. Their association gives rise to a hybrid construct combining biological and material components that can be specifically committed. The comprehension of cellular and molecular mechanisms of cartilage repair and the use of biomaterials in combination with chondrocytes or mesenchymal stem cells in the treatment of cartilage defects has opened a new era of therapeutical strategies. Recently, their applicability in the treatment of early lesions in osteoarthritis is under investigation. To obtain new information on the behaviour of chondrocytes and mesenchymal stem cells grown on a hyaluronan derivative scaffold (Hyaff-11) already used in cartilage repair, we analysed a series of molecules expressed by these cells by Real-Time RT-PCR and immunohistochemical analyses. The data obtained with this work showed that this biomaterial is able to reduce the expression of some catabolic molecules by human chondrocytes and provide a good environment to support the differentiation of mesenchymal stem cells in chondrogenic sense. These observations confirm Hyaff-11 as a suitable scaffold both for chondrocytes and mesenchymal stem cells for the treatment of articular cartilage defects.

Adult↗

PubMed2003

Endovascular restorative neurosurgery: a novel concept for molecular and cellular therapy of the nervous system.

The amalgam of molecular biology and neurosurgery offers immense promise for neurorestoration and the management of neurodegenerative deficiencies, developmental disorders, neoplasms, stroke, and trauma. This article summarizes present strategies for and impediments to gene therapy and stem cell therapy of the central nervous system and advances the concept of a potential new approach, namely endovascular restorative neurosurgery. The objectives of gene transfer to the central nervous system are efficient transfection of host cells, selective sustained expression of the transgene, and lack of toxicity or immune excitation. The requisite elements of this process are the identification of candidate diseases, the construction of vehicles for gene transfer, regulated expression, and physical delivery. In the selection of target disorders, the underlying genetic events to be overcome, as well as their spatial and temporal distributions, must be considered. These factors determine the requirements for the physical dispersal of the transgene, the duration of transgene expression, and the quantity of transgene product needed to abrogate the disease phenotype. Vehicles for conveying the transgene to the central nervous system include viral vectors (retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, and herpes simplex virus), liposomes, and genetically engineered cells, including neural stem cells. Delivery of the transgene into the brain presents several challenges, including limited and potentially risky access through the cranium, sensitivity to volumetric changes, restricted diffusion, and the blood-brain barrier. Genetic or cellular therapeutic agents may be injected directly into the brain parenchyma (via stereotaxy or craniotomy), into the cerebrospinal fluid (in the ventricles or cisterns), or into the bloodstream (intravenously or intra-arterially). The advantages of the endovascular route include the potential for widespread distribution, the ability to deliver large volumes, limited perturbation of neural tissue, and the feasibility of repeated administration.

Animals↗

PubMed2005

Obstructive nephropathy: insights from genetically engineered animals.

Congenital obstructive nephropathy is the primary cause for end-stage renal disease (ESRD) in children. An increasingly used animal model of obstructive nephropathy is unilateral ureteral obstruction (UUO). This model mimics, in an accelerated manner, the different stages of obstructive nephropathy leading to tubulointerstitial fibrosis: cellular infiltration, tubular proliferation and apoptosis, epithelial-mesenchymal transition (EMT), (myo)fibroblast accumulation, increased extracellular matrix (ECM) deposition, and tubular atrophy. During the last decade genetically modified animals are increasingly used to study the development of obstructive nephropathy. Although the use of these animals (mainly knockouts) has highlighted some pitfalls of this approach (compensation by closely related gene products, absence of temporal knockouts) it has brought important information about the role of specific gene-products in the pathogenesis of obstructive nephropathy. Besides confirming the important pathologic role for angiotensin II (Ang II) and transforming growth factor-beta (TGF-beta) in obstructive nephropathy, these animals have shown the complexity of the development of tubulointerstitial fibrosis involving a large number of closely functionally related molecules. More interestingly, the use of these animals has led to the discovery of unexpected and contradictory roles (both potentially pro- and antifibrotic) for Ang II, for ECM degrading enzymes matrix metalloproteinase 9 (MMP-9) and tissue plasminogen activators (PAs), for plasminogen activator inhibitor 1 (PAI-1), and for the adhesion molecule osteopontin (OPN) in obstructive nephropathy. Further use of these animals, especially in combination with pharmacologic tools, should help to better identify potential antifibrotic strategies in obstructive nephropathy.

Animals↗

PubMed2004

Spectral and lifetime fluorescence imaging microscopies: new modalities of multiphoton microscopy applied to tissue or cell engineering.

Spectral and multiphoton imaging is the preferred approach for non-invasive study allowing deeper penetration to image molecular processes in living cells. But currently available fluorescence microscopic techniques based on fluorescence intensity, such as confocal or multiphoton excitation, cannot provide detailed quantitative information about the dynamic of complex cellular structure (molecular interaction). Due to the variation of the probe concentration, photostability, cross-talking, its effects cannot be distinguished in simple intensity images. Therefore, Time Resolved fluorescence image is required to investigate molecular interactions in biological systems. Fluorescence lifetimes are generally absolute, sensitive to environment, independent of the concentration of the probe and allow the use of probes with overlapping spectra but that not have the same fluorescence lifetime. In this work, we present the possibilities that are opened up by Fluorescence Lifetime Imaging Microscopy, firstly to collect images based on fluorescence lifetime contrast of GFP variants used as a reporter of gene expression in chondrocytes and secondly, to measure molecular proximity in erythrocyte (glycophorin/membrane) by Fluorescence Resonance Energy Transfer (FLIM-FRET).

Erythrocytes↗