Green Fluorescent Protein Marker Research Articles

Vascular-specific expression of GUS and GFP reporter genes in transgenic grapevine (Vitis vinifera L. cv. Albariño) conferred by the EgCCR promoter of Eucalyptus gunnii

In the view of the economic importance of grapevine and the increasing threaten represented by vascular diseases, transgenic grapevine with enhanced tolerance could represent an attractive opportunity. Hitherto, constitutive promoters have been used generally to study the effects of transgene expression in grapevine. Given the fact that constitutive gene expression may be harmful to the host plant, affecting plant growth and development, the use of tissue -specific promoters restricting gene expression to tissues of interest and at given developmental stages could be more appropriate. For this purpose, we decided to study in grapevine the activity of the Eucalyptus gunnii CCR promoter that was previously reported to be vascular-preferential. We transformed grapevine with the "Sonication assisted Agrobacterium-mediated transformation" (SAAT) method and a construct where both GUS and GFP (green fluorescent protein) marker genes were under control of the EgCCR promoter. High GUS and GFP activities were found to be associated with the newly formed vascular tissues in stems, leaves and petioles of transformed grapevine, suggesting a preferential activity of the EgCCR promoter in the vascular tissues of grapevine. These results suggest the tissue-specificity of this promoter from eucalyptus is conserved in grapevine and that it could be used to drive expression of defense genes in order to enhance resistance against vascular pathogens.

嗅組織と再生医療

Regeneration medicine is divided mainly into differentiation-induced and cell replenishment therapy. Bone marrow includes stem cell populations such as hematopoietic, mesenchymal and so on. These stem cells are shown experimentally and clinically to be pluripotent. It remains unclear, however, whether bone-marrow-derived cells differentiate into olfactory neurons and are engrafted as olfactory stem cells into olfactory tissue. We transplanted bone marrow cells of green fluorescence protein (GFP) mice into lethally irradiated recipients. Double immunostaining for GFP and olfactory marker protein or cytokeratin showed the possibility that bone-marrow-derived cell population could do as hoped. Because the engraftment rates of this study were low far from a practical level, methimazole inducing selectively apoptosis into olfactory tissue and granulocyte colony-stimulating factor known to mobilize stem cells from bone marrow into circulation were added to a series of experiments, which increased engraftment rates. Whether bone-marrow-derived cells or mesenchymal stem cells differentiate into olfactory cells remains controversial, necessitating the evaluation of regeneration medicine using such cells in further olfactory disorder studies.

O22. Cardiovascular derivatives of embryonic stem cells in cardiac repair and drug discovery

Derivation of heart and vascular endothelial cells from human pluripotent stem cells (embryonic stem cells or HESCs and induced pluripotency stem cells or hiPS cells) is an area of growing interest both as a route to cell therapy for the heart and as a platform for drug discovery and toxicity. Understanding the underlying developmental mechanisms that control differentiation of pluripotent cells to cardiac progenitors and their derivatives and mimicking these in defined culture conditions in vitro is now essential for moving the field forward. Culture conditions have now been sufficiently refined that cardiomyocyte and vascular differentiation is a fairly efficient and reproducible process. Genetically marked HESCs have been produced in which expression of the green fluorescent protein marker is expressed ubiquitously or driven by specific lineage markers. We have used these tagged lines to trace cardiomyocytes following transplantation into a mouse heart after myocardial infarction and select the progenitors of cardiomyocytes, endothelial cells and smooth muscle cells. Long term survival of the cells and integration into the host heart has been observed and early improvements in cardiac function but these are not sustained. Cardiac repair using stem cell derived cardiomyocytes will likely require more than efficient cardiomyocytes production. More immediate applications of hESC- and hiPSC derived cardiomyocytes and vascular endothelial cells in drug discovery and disease are now close to implementation. Results of these studies, in particular drug responses of hESC-derived cardiomyocytes and an hiPSC model for vascular disease in which Thalidomide has a therapeutic effect, will be shown.

Gene delivery in the equine cornea: a novel therapeutic strategy

To determine if hybrid adeno-associated virus serotype 2/5 (AAV5) vector can effectively deliver foreign genes into the equine cornea without causing adverse side effects. The aims of this study were to: (i) evaluate efficacy of AAV5 to deliver therapeutic genes into equine corneal fibroblasts (ECFs) using enhanced green fluorescent protein (EGFP) marker gene, and (ii) establish the safety of AAV5 vector for equine corneal gene therapy. Primary ECF cultures were harvested from healthy donor equine corneas. Cultures were maintained at 37°C in humidified atmosphere with 5% CO(2). AAV5 vector expressing EGFP under control of hybrid cytomegalovirus + chicken β-actin promoter was applied topically to ECF. Expression of delivered EGFP gene in ECF was quantified using fluorescent microscopy. Using fluorescent staining, the total number of cells and transduction efficiency of tested AAV vector was determined. Phase contrast microscopy, trypan blue and TUNEL assays were used to determine toxicity and safety of AAV5 for ECFs. Topical AAV5 application successfully transduced significant numbers of ECFs. Transduction efficiency was 13.1%. Tested AAV5 vector did not cause phenotype change or significant cell death and cell viability was maintained. Tested AAV5 vector is effective and safe for gene therapy in ECFs in vitro.

Robust In Vivo Transduction of Nervous System and Neural Stem Cells by Early Gestational Intra Amniotic Gene Transfer Using Lentiviral Vector

Presently, in vivo methods to efficiently and broadly transduce all major cell types throughout both the central (CNS) and peripheral adult nervous system (PNS) are lacking. In this study, we hypothesized that during early fetal development neural cell populations, including neural stem cells (NSCs), may be accessible for gene transfer via the open neural groove. To test this hypothesis, we injected lentiviral vectors encoding a green fluorescent protein (GFP) marker gene into the murine amniotic cavity at embryonic day 8. This method (i) efficiently and stably transduced the entire nervous system for at least 80% of the lifespan of the mice, (ii) transduced all major neural cell types, and (iii) transduced adult NSCs of the subventricular zone (SVZ) and subgranular zones (SGZs). This simple approach has broad applications for the study of gene function in nervous system development and adult NSCs and may have future clinical applications for treatment of genetic disorders of the nervous system.

Notch Signaling: Filopodia Dynamics Confer Robustness

Though usually thought of as mediating communication between adjacent cells, Notch-Delta signaling can take place over a longer range through cellular processes known as 'filopodia'. A recent study shows how the dynamics of filopodia can confer robustness to Notch-Delta dependent patterning.

A Novel Gene,ROA, Is Required for Normal Morphogenesis and Discharge of Ascospores in Gibberella zeae

Head blight, caused by Gibberella zeae, is a significant disease among cereal crops, including wheat, barley, and rice, due to contamination of grain with mycotoxins. G. zeae is spread by ascospores forcibly discharged from sexual fruiting bodies forming on crop residues. In this study, we characterized a novel gene, ROA, which is required for normal sexual development. Deletion of ROA (Δroa) resulted in an abnormal size and shape of asci and ascospores but did not affect vegetative growth. The Δroa mutation triggered round ascospores and insufficient cell division after spore delimitation. The asci of the Δroa strain discharged fewer ascospores from the perithecia but achieved a greater dispersal distance than those of the wild-type strain. Turgor pressure within the asci was calculated through the analysis of osmolytes in the epiplasmic fluid. Deletion of the ROA gene appeared to increase turgor pressure in the mutant asci. The higher turgor pressure of the Δroa mutant asci and the mutant spore shape contributed to the longer distance dispersal. When the Δroa mutant was outcrossed with a Δmat1-2 mutant, a strain that contains a green fluorescence protein (GFP) marker in place of the MAT1-2 gene, unusual phenotypic segregation occurred. The ratio of GFP to non-GFP segregation was 1:1; however, all eight spores had the same shape. Taken together, the results of this study suggest that ROA plays multiple roles in maintaining the proper morphology and discharge of ascospores in G. zeae.

Complete regression of human malignant mesothelioma xenografts following local injection of midkine promoter‐driven oncolytic adenovirus

Malignant mesothelioma is a highly aggressive tumor with poor prognosis. We hypothesized that the tumor-specific midkine (Mdk) promoter could confer transcriptional targeting to oncolytic adenoviruses for effective treatment of malignant mesothelioma. We analysed Mdk expression by quantitative reverse transcription-polymerase chain reaction in six human mesothelioma cell lines, and tested Mdk promoter activity by luciferase reporter assay. On the basis of these data, we constructed a replication-selective oncolytic adenovirus designated AdMdk-E1-iresTK. This virus contains a Mdk promoter-driven adenoviral E1 gene and herpes simplex virus-thymidine kinase (TK) suicide gene and cytomagalovirus promoter-driven enhanced green fluorescent protein marker gene. Selectivity of viral replication and cytolysis were characterized in normal versus mesothelioma cells in vitro, and intratumoral spread and antitumor efficacy were investigated in vivo. Mdk promoter activity was restricted in normal cells, but highly activated in mesothelioma cell lines. AdMdk-E1-iresTK was seen to efficiently replicate, produce viral progeny and spread in multiple mesothelioma cell lines. Lytic spread of AdMdk-E1-iresTK mediated the efficient killing of these mesothelioma cells, and its in vitro cytocidal effect was significantly enhanced by treatment with the prodrug, ganciclovir. Intratumoral injection of AdMdk-E1-iresTK caused complete regression of MESO4 and MSTO human mesothelioma xenografts in athymic mice. In vivo fluorescence imaging demonstrated intratumoral spread of AdMdk-E1-iresTK-derived signals, which vanished after tumor eradication. These data indicate that transcriptional targeting of viral replication by the Mdk promoter represents a promising general strategy for oncolytic virotherapy of cancers with up-regulated Mdk expression, including malignant mesothelioma.

Corneal myofibroblast generation from bone marrow-derived cells

The purpose of this study was to determine whether bone marrow-derived cells can differentiate into myofibroblasts, as defined by alpha-smooth muscle actin (SMA) expression, that arise in the corneal stroma after irregular phototherapeutic keratectomy and whose presence within the cornea is associated with corneal stromal haze. C57BL/6J-GFP chimeric mice were generated through bone marrow transplantation from donor mice that expressed enhanced green fluorescent protein (GFP) in a high proportion of their bone marrow-derived cells. Twenty-four GFP chimeric mice underwent haze-generating corneal epithelial scrape followed by irregular phototherapeutic keratectomy (PTK) with an excimer laser in one eye. Mice were euthanized at 2 weeks or 4 weeks after PTK and the treated and control contralateral eyes were removed and cryo-preserved for sectioning for immunocytochemistry. Double immunocytochemistry for GFP and myofibroblast marker alpha-smooth muscle actin (SMA) were performed and the number of SMA+GFP+, SMA+GFP−, SMA−GFP+ and SMA−GFP− cells, as well as the number of DAPI+ cell nuclei, per 400× field of stroma was determined in the central, mid-peripheral and peri-limbal cornea. In this mouse model, there were no SMA+ cells and only a few GFP+ cells detected in unwounded control corneas. No SMA+ cells were detected in the stroma at two weeks after irregular PTK, even though there were numerous GFP+ cells present. At 4 weeks after irregular PTK, all corneas developed mild to moderately severe corneal haze. In each of the three regions of the corneas examined, there were on average more than 9× more SMA+GFP+ than SMA+GFP− myofibroblasts. This difference was significant ( p < 0.01). There were significantly more ( p < 0.01) SMA−GFP+ cells, which likely include inflammatory cells, than SMA+GFP+ or SMA+GFP− cells, although SMA−GFP− cells represent the largest population of cells in the corneas. In this mouse model, the majority of myofibroblasts developed from bone marrow-derived cells. It is possible that all myofibroblasts in these animals developed from bone marrow-derived cells since mouse chimeras produced using this method had only 60–95% of bone marrow-derived cells that were GFP+ and it is not possible to achieve 100% chimerization. This model, therefore, cannot exclude the possibility of myofibroblasts also developed from keratocytes and/or corneal fibroblasts.

Enrichment of Testicular Gonocytes and Genetic Modification Using Lentiviral Transduction in Pigs1

Gonocytes are long-lived primary germ cells that reside in the center of seminiferous cords until differentiation into spermatogonia that drive spermatogenesis. In pigs, gonocytes have research value in the production of transgenic offspring through germline modification and transplantation. However, the rarity of pig gonocytes has raised the need for an efficient isolation method. Therefore, in this study we use components of extracellular matrix, laminin, fibronectin, and collagen type IV and their derivative, gelatin, to establish a negative selection system for functionally viable gonocytes in neonatal pig. We then demonstrate functional analysis with genetic modification using lentiviral transduction and successfully transplant the donor gonocytes, which colonized the seminiferous tubules of the recipient mouse. The most effective selection method was established by sequential use of laminin and gelatin, in which the purity of gonocytes was 80% and the recovery rate of gonocytes was 78%. The selected gonocytes were labeled with fluorescent dye PKH26 and transplanted into busulfan-treated immunodeficient mouse testes. The fluorescent gonocytes colonized the recipient testes, and the resultant germ cell colonies were visible up to 4 mo after transplantation. When gonocytes were transplanted after transduction with an enhanced green fluorescent protein marker gene using lentiviral vectors, the transduced germ cell colonies were visible up to 6 mo and displayed an estimated transduction efficiency of 11.1%. These results can be applied and extended to isolate and enrich gonocytes of other species for in vitro and in vivo studies and to assist in genetic modification of male germline stem cells of livestock species.

Cytokine stimulation and the choice of promoter are critical factors for the efficient transduction of mouse T cells with HIV‐1 vectors

HIV-1 fails to successfully infect mouse T cells as a result of several blocks in the viral replication cycle. We investigated whether this also impacted on the use of HIV-1 derived lentiviral vectors for stable gene transfer into mouse T cells. Freshly isolated primary mouse T cells were immediately mixed with lentiviral vectors encoding an enhanced green fluorescent protein marker gene and transduction frequency was determined after 5 days of culture. Optimal transduction required both mouse T cell activation and cytokine support. Furthermore, transduction was also dependent upon the promoter chosen, with the rank order of potency being PGK > EF1 > SFFV > CMV. HIV-1 lentiviral vectors also efficiently transduced cytokine-stimulated T cells (in the absence of antibody driven T cell activation), albeit with a lower level of transgene expression compared to fully-activated T cells. The present study demonstrates that primary mouse T cells can be efficiently transduced with HIV-1 lentiviral vectors, opening up prospects for their use in mouse models of gene-modified adoptive cellular therapy.

Silencing induced by inverted repeat constructs in protoplasts of Nicotiana benthamiana

Double-stranded RNA interference can be used to silence gene expression in various organisms. Sustained RNAi-mediated gene silencing is typically triggered by hairpin RNAs (hpRNAs) generated by the transcription of inverted repeat (IR) DNA constructs. In this paper, we describe a transient expression assay that uses a green fluorescent protein marker gene (GFP) fused to the coat protein gene (CP) of papaya ring spot virus. This yields a nuclear and cytomembrane localized protein expression vector that can be used to monitor the silencing efficiency of inverted repeats. An ihpRNA was derived from a 215 nt IR segment of the 3′ end of the CP gene and recombined into the pHellsgate12 vector. We also recombined the 861 nt CP into the destination vectors pHellsgate12 and pWatergate using the GatewayTM technology. Nicotiana benthamiana protoplasts were co-transfected with (1) pGA, (2) pGA + pHellsgate12-CPIR, (3) pGA + pWatergate-CPIR, and (4) pGA + pHellsgate12-CP215IR. Expression of the GFP fusion protein decreased by 77.7, 75.4, and 65.6% for the three constructs. Transfection with (2) and (3) yielded a silencing efficiency significantly higher than that of the (4) (P < 0.05), as measured by fluorescence intensity upon co-transfection, using the vector pGA-DsRed as an internal control. Depletions of 90.7, 91.5, and 87.5% of the CP transcript were observed by RT-PCR in protoplasts co-transfected with the (1) pGA, (2) pGA + pHellsgate12-CPIR, and (3) pGA + pWatergate-CPIR, (4) pGA + pHellsgate12-CP215IR. Transient RNAi depletion of the target polypeptide was also detected by western blot analysis. Short RNA fragments complementary to the p6a and p7a antisense probes were detected by Ribonuclease Protection Assays.

Identification of virus resistant tumor cell subpopulations in three-dimensional uveal melanoma cultures

To better understand melanoma resistance to herpes simplex virus type 1 (HSV-1)-mediated oncolysis, traditional two-dimensional (2D) cultures and extracellular matrix (ECM) containing three-dimensional (3D) cultures of OCM1 and C918 uveal melanoma cells were infected with an HSV-1 strain that expresses the green fluorescent protein (GFP) marker during replication. Although 2D cultures were completely destroyed within a few days of HSV-1 inoculation, viable GFP-negative tumor cells remained detectable in 3D cultures for several weeks. Tumor cells with increased resistance to HSV-1 included cells that formed vasculogenic mimicry patterns and multicellular spheroids and cells that invaded Matrigel individually. Mechanisms of tumor resistance against HSV-1 in the 3D environment included impaired virus spread in the ECM and ECM-mediated inhibition of viral replication after viral entry into tumor cells. Observations also suggested that HSV-1 established quiescent infection in some tumor cells present in multicellular spheroids and that this could revert to productive viral infection when the tumor growth pattern changed. These findings indicate that 3D tumor cell cultures can be used to identify distinct tumor cell populations with increased resistance to HSV-1 and to explore mechanisms of ECM-mediated tumor resistance to oncolytic virotherapy.

Regeneration of transgenic citrus plants from the trimmed shoot/root region of etiolated seedlings

Transformation and high efficient regeneration of transgenic plants from the trimmed etiolated shoot/root region (TESRR) of Anliucheng sweet orange [Citrus sinensis (L.) Osb.] seedling was reported. A visual green fluorescent protein (GFP) marker gene was introduced to evaluate transformation efficiency by using the explants from TESRR and epicotyls. The transformation protocol was: infection 20 min, co-culture 3 d, selection culture 30 d, and rooting 15 d. Out of a total of 288 sprouted shoots obtained from TESRR, 34 shoots (11.8 %) yielded GFP expression. In contrast, only 2 (3.0 %) of the 67 sprouted shoots from epicotyl transformation yielded GFP expression. In all plants showing the green fluorescence an expected 500 bp GFP fragment was proved by PCR analysis. Southern blot analysis further confirmed the integration of GFP gene into citrus genome. Transgenic plantlets were obtained within 80 d using the TESRR, compared within 150 d by using epicotyls.

Green fluorescent protein (GFP) as a vital marker for studying the interaction of Phytophthora sojae and soybean

Transgenic Phytophthora sojae strains that produce green fluorescent protein (GFP) were obtained after stable DNA integration using the Hsp70 promoter and the Ham34 terminator of Bremia lactucae. The expression of GFP during different developmental stages of P. sojae was observed using fluorescent microscopy. Based on this reporter system, the histopathologic events caused by the pathogen in soybean leaves, hypocotyls and roots were monitored. Meanwhile, the difference in resistance between different soybean cultivars against P. sojae was analyzed microscopically in roots. The results indicate that GFP can be stably expressed in zoosporangia, zoospores, cysts, hyphae and oospores of P. sojae. Using the GFP marker, the infecting pathogens in leaves, hypocotyls and roots of host could be distinctly visualized. The germ tube length of cysts germinating on the roots of resistant cultivar Nannong 8848 was longer than that on the roots of susceptible cultivar Hefeng 35. These results show for the first time that this eukaryotic reporter can be used in P. sojae as a stable and vital marker, allowing the study of genetics of this hemibiotrophic pathogen.

Remission of Type 1 Diabetes after Anti-CD3 Antibody Treatment and Transplantation of Embryonic Pancreatic Precursors

Type 1 diabetes (T1D) mellitus is characterized by progressive autoimmune destruction of insulin producing beta-cells of the pancreatic islets of Langerhans. Cure of the disease will require control of autoimmunity to halt the destruction of beta-cells in the pancreas and restoration of beta-cell mass. We have built on the success of preclinical and clinical trials of anti-CD3 antibody treatment in modulating the immune response of T1D by the induction of tolerance and combined this treatment, using the nonobese diabetic mouse model, with a transplantation approach using fetal pancreatic anlagen as a source of beta-cell precursor or progenitor cells. Here we report that transplantation of pancreatic anlagen into diabetic nonobese diabetic mice rendered tolerant to the autoimmune process by treatment with anti-CD3 antibody resulted in long-term recovery from diabetes with restored metabolic control. Using a green fluorescent protein marker that made it possible to unequivocally identify the cells derived from the transplanted tissue, we show that the transplanted anlagen cells migrate to the host pancreas and provide a major source of insulin leading to restoration of normal glucose tolerance. Our results contrast with other studies that showed restoration of endogenous islets after infusion of spleen cells in mice treated with Freund's complete adjuvant and suggest that pancreatic fetal tissue has a tropism for the pancreatic site. This study suggests a novel mechanism of beta-cell restoration by the migration of precursor cells or their progeny to the host pancreas and highlights the feasibility of using pancreatic precursors in combination with immune modulation as a treatment to effect long-term remission of T1D.

A bacterial signal peptide is functional in plants and directs proteins to the secretory pathway

The Escherichia coli heat-labile enterotoxin B subunit (LT-B) has been used as a model antigen for the production of plant-derived high-valued proteins in maize. LT-B with its native signal peptide (BSP) has been shown to accumulate in starch granules of transgenic maize kernels. To elucidate the targeting properties of the bacterial LT-B protein and BSP in plant systems, the subcellular localization of visual marker green fluorescent protein (GFP) fused to LT-B and various combinations of signal peptides was examined in Arabidopsis protoplasts and transgenic maize. Biochemical analysis indicates that the LT-B::GFP fusion proteins can assemble and fold properly retaining both the antigenicity of LT-B and the fluorescing properties of GFP. Maize kernel fractionation revealed that transgenic lines carrying BSP result in recombinant protein association with fibre and starch fractions. Confocal microscopy analysis indicates that the fusion proteins accumulate in the endomembrane system of plant cells in a signal peptide-dependent fashion. This is the first report providing evidence of the ability of a bacterial signal peptide to target proteins to the plant secretory pathway. The results provide important insights for further understanding the heterologous protein trafficking mechanisms and for developing effective strategies in molecular farming.

The Xanthomonas campestris pv. vesicatoria Type III Effector Protein XopJ Inhibits Protein Secretion: Evidence for Interference with Cell Wall–Associated Defense Responses

The phytopathogenic bacterium Xanthomonas campestris pv. vesicatoria uses the type III secretion system (T3SS) to inject effector proteins into cells of its Solanaceous host plants. It is generally assumed that these effectors manipulate host pathways to favor bacterial replication and survival. However, the molecular mechanisms by which type III effectors suppress host defense responses are far from being understood. Based on sequence similarity, Xanthomonas outer protein J (XopJ) is a member of the YopJ/AvrRxv family of SUMO peptidases and acetyltranferases, although its biochemical activity has not yet been demonstrated. Confocal laser scanning microscopy revealed that green fluorescent protein (GFP) fusions of XopJ are targeted to the plasma membrane when expressed in plant cells, which most likely involves N-myristoylation. In contrast to a XopJ(C235A) mutant disrupted in the catalytic triad sequence, the wild-type effector GFP fusion protein was also localized in vesicle-like structures colocalizing together with a Golgi marker protein, suggesting an effect of XopJ on vesicle trafficking. To explore an effect of XopJ on protein secretion, we used a GFP-based secretion assay. When a secreted (sec)GFP marker was coexpressed with XopJ in leaves of Nicotiana benthamiana, GFP fluorescence was retained in reticulate structures. In contrast, in plant cells expressing secGFP alone or along with the XopJ(C235A) mutant, no GFP fluorescence accumulated within the cells. Moreover, coexpressing secGFP together with XopJ led to a reduced accumulation of secGFP within the apoplastic fluid of N. benthamiana leaves, further showing that XopJ affects protein secretion. Transgenic expression of XopJ in Arabidopsis suppressed callose deposition elicited by a T3SS-negative mutant of Pseudomonas syringae pv. tomato DC3000. A role of XopJ in the inhibition of cell wall-based defense responses is discussed.

Grafting the Olfactory Epithelium to the Olfactory Bulb

Impaired olfactory function leads to a decrease in the quality of life for many patients. Surgical treatment options are limited, especially for those suffering from hyposmia or anosmia after posttraumatic injury to the olfactory nerves. Stem cells located in the olfactory epithelium (OE) have the capacity to grow new neurons, making the OE an ideal candidate for restorative tissue grafting. This study was performed to determine if strips of OE survive transplantation directly to the olfactory bulb (OB). Transgenic mice, expressing a green fluorescent protein (GFP), were used to obtain the donor graft tissue. Strips of OE from GFP donor mice were transplanted directly to sites in the OB and cerebral cortex (CC; control sites) of wild-type mice. Graft survival rates at 30 days were determined for transplant sites in the OB and CC. Strips of OE from transgenic mice survived transplantation to the OB and continued to express the GFP marker protein. The 30-day survival rate in the OB (83%, 5 of 6 grafts) was the same as in the CC (10 of 12 grafts). The morphology of the graft revealed characteristics found in normal OE. We showed that strips of OE can be successfully grafted to both the OB and CC. Grafts of the OE, if strategically positioned on the ventral surface of the bulb and given access to the nasal cavity, could provide the basis for new surgical treatments to restore olfactory function.

The Use of Short Tandem Repeat Profiling to Characterize Human Bladder Cancer Cell Lines

Cross-contamination of cell lines is a serious but often unrecognized problem. We describe the authentication of a panel of transitional cell carcinoma cell lines using the short tandem repeat profiling technique to detect cross-contamination. Genomic DNA was isolated from UM-UC-1, UM-UC-2, UM-UC-3 (ATCC), UM-UC-6, UM-UC-9, UM-UC-10, UM-UC-11, UM-UC-13, UM-UC-14, UM-UC-16, T24 and KU7 cell lines. Short tandem repeat loci (D3S1358, D16S539, vWA, FGA, TH01, TPOX, CSF1PO, D5S818, D13S317 and D7S820) and a segment of the X-Y homologous gene amelogenin were co-amplified by polymerase chain reaction. Profiling was done using POP-4TM performance optimized polymer 4 (Applied Biosystems) with an ABI Prism 310 genetic analyzer. DNA sequencing of TP53 and immunohistochemistry for p53 were performed in UM-UC-3 and UM-UC-3-GFP. All cell lines had a unique short tandem repeat profile except UM-UC-2 and T24, which were virtually identical. T24 short tandem repeat profiles matched those of early passage number UM-UC-2. Stable transfection of the green fluorescence protein marker gene did not alter UM-UC-6, UM-UC-14 or KU7 profiles. However, the short tandem repeat profile for UM-UC-3-GFP was different from that of UM-UC-3. DNA sequencing showed a difference in TP53 between UM-UC-3 and UM-UC-3-GFP, confirming that UM-UC-3-GFP is not derived from UM-UC-3. Short tandem repeat profiling provides a unique genetic signature of human cell lines that does not significantly change with passage or green fluorescence protein transduction. Using short tandem repeat profiling we noted that the cell line UM-UC-2 is T24. DNA fingerprinting using short tandem repeat profiling is an easy and reliable tool that can be used to verify cell lines.