Detectable Reporter Gene Expression Research Articles

Measurement of Secreted Embryonic Alkaline Phosphatase.

Secreted reporters have been demonstrated to be simple and useful tools for analyzing transcriptional regulation in mammalian cells. The distinctive feature of these assays is the ability to detect reporter gene expression in the culture supernatant without affecting the cell physiology or leading to cell lysis, which allows repeated experimentation and sampling of the culture medium using the same cell cultures. Secreted embryonic alkaline phosphatase (SEAP) is one of the most widely used reporter, which can be easily detected using colorimetry following incubation with a substrate, such as p-nitrophenol phosphate. In this report, we present detailed procedures for detection and quantification of the SEAP reporter. We believe that this step-by-step protocol can be easily used by researchers to monitor and measure molecular genetic events in a variety of mammalian cells due to its simplicity and ease of handling. Graphical abstract Schematic overview of the workflow described in this protocol.

Methods and Sensors For Functional Genomic Studies of Cell‐Cycle Transitions in Single Cells

Our understanding of the genetic control mechanisms governing the cell cycle has relied heavily on methods that measure the aggregate state of a population of cells. While instrumental in shaping our fundamental understanding of much of what we know about how the cell cycle is regulated, these approaches also mask the signaling and genetic signatures of rare subpopulations. Quiescent (G0) or very slow dividing (SD) cells are two such related populations. Our data and that of others show that the majority of single clonal MCF7 breast cancer cells cultured at subconfluency can complete a full round of cell division in as little as 24h, however subpopulations within that culture (1–5%) can require over 100h to complete the cell cycle. Moreover, it is not clear how these phenotypes are transferred to daughter cells. Understanding the proteins and networks that control G0 or SD cells may lead to the identification of new drug targets for aging or tissue regeneration. This study establishes an integrated computational, imaging and molecular pipeline that enables the phenotyping of G0 or SD cultured cells within a population. We define imaging parameters and open source R scripts to generate and analyze time‐lapse images to track and quantify cell cycle transition in individual cells expressing a genetically encoded cell cycle reporter system (Ki67p‐FUCCI). We report how these cells can be used to identify transcriptomic signatures of G0, G1 and G2 phase cells within a population. Additionally, we present single‐cell microinjection parameters to optimize single cell transfection by direct injection of mRNA in MCF7 cells with detectable reporter gene expression (GFP) within 5h of microinjection. These findings coupled with the possibility of targeting several hundreds of single cells within a field of view while simultaneously conducting 4 channel time‐lapse microscopy dramatically increase throughput and sensitivity over conventional methods for functional genomics studies to identify new genes that control cell cycle transitions.

Double‐tagged fluorescent bacterial bioreporter for the study of polycyclic aromatic hydrocarbon diffusion and bioavailability

Bacterial degradation of polycyclic aromatic hydrocarbons (PAHs), ubiquitous contaminants from oil and coal, is typically limited by poor accessibility of the contaminant to the bacteria. In order to measure PAH availability in complex systems, we designed a number of diffusion-based assays with a double-tagged bacterial reporter strain Burkholderia sartisoli RP037-mChe. The reporter strain is capable of mineralizing phenanthrene (PHE) and induces the expression of enhanced green fluorescent protein (eGFP) as a function of the PAH flux to the cell. At the same time, it produces a second autofluorescent protein (mCherry) in constitutive manner. Quantitative epifluorescence imaging was deployed in order to record reporter signals as a function of PAH availability. The reporter strain expressed eGFP proportionally to dosages of naphthalene or PHE in batch liquid cultures. To detect PAH diffusion from solid materials the reporter cells were embedded in 2 cm-sized agarose gel patches, and fluorescence was recorded over time for both markers as a function of distance to the PAH source. eGFP fluorescence gradients measured on known amounts of naphthalene or PHE served as calibration for quantifying PAH availability from contaminated soils. To detect reporter gene expression at even smaller diffusion distances, we mixed and immobilized cells with contaminated soils in an agarose gel. eGFP fluorescence measurements confirmed gel patch diffusion results that exposure to 2-3 mg lampblack soil gave four times higher expression than to material contaminated with 10 or 1 (mg PHE) g(-1).

Detectable reporter gene expression following transduction of adenovirus and adeno‐associated virus serotype 2 vectors within full‐thickness osteoarthritic and unaffected canine cartilage in vitro and unaffected guinea pig cartilage in vivo

This study quantified and compared the transduction efficiencies of adenoviral (Ad), Arg-Gly-Asp (RGD)-modified Ad, adeno-associated viral serotype 2 (AAV2), and self-complementary AAV2 (scAAV2) vectors within full-thickness osteoarthritic (OA) and unaffected canine cartilage explants in vitro. Intraarticular administration of Ad and scAAV2 vectors was performed to determine the ability of these vectors to transduce unaffected guinea pig cartilage in vivo. Following explant exposure to vector treatment or control, the onset and surface distribution of reporter gene expression was monitored daily with fluorescent microscopy. At termination, explants were divided: one half was digested for analysis using flow cytometry; the remaining portion was used for histology and immunohistochemistry (IHC). Intact articular joints were collected for real-time RT-PCR and IHC to detect reporter gene expression following injection of selected vectors. Ad vector transduced focal areas along the perimeters of explants; the remaining vectors transduced chondrocytes across 100% of the surface. Greater mean transduction efficiencies were found with both AAV2 vectors as compared to the Ad vector (p < or = 0.026). Ad and Ad-RGD vectors transduced only superficial chondrocytes of OA and unaffected cartilage. Uniform reporter gene expression from AAV2 and scAAV2 was detected in the tangential and transitional zones of OA cartilage, but not deeper zones. AAV2 and scAAV2 vectors achieved partial and full-thickness transduction of unaffected cartilage. In vivo work revealed that scAAV2 vector, but not Ad vector, transduced deeper zones of cartilage and menisci. This study demonstrates that AAV2 and scAAV2 are reliable vectors for use in cartilage in vitro and in vivo.

Lentivirus-mediated gene transfer to the rat, ovine and human cornea

Gene therapy of the cornea shows promise for modulating corneal transplant rejection but the most appropriate vector for gene transfer has yet to be determined. We investigated a lentiviral vector (LV) for its ability to transduce corneal endothelium. A lentivector expressing enhanced yellow fluorescent protein (eYFP) under the control of the Simian virus type 40 early promoter (LV-SV40-eYFP) transduced 80-90% of rat, ovine and human corneal endothelial cells as detected by fluorescence microscopy. The kinetics of gene expression varied among species, with ovine corneal endothelium showing a relative delay in detectable reporter gene expression compared with the rat or human corneal endothelium. Vectors containing the myeloproliferative sarcoma virus promoter or the phosphoglycerate kinase promoter were not significantly more effective than LV-SV40-eYFP. The stability of eYFP expression in rat and ovine corneas following ex vivo transduction of the donor cornea was assessed following orthotopic corneal transplantation. Following transduction ex vivo, eYFP expression was maintained in corneal endothelial cells for at least 28 days after corneal transplantation in the sheep and >60 days in the rat. Thus, rat, ovine and human corneal endothelial cells were efficiently transduced by the LV, and gene expression appeared stable over weeks in vivo.

Myeloid Lineage-Restricted Progenitors Contribute to Vascular Endothelium.

Endothelial progenitors of hematopoietic origin have a role in promoting recovery from vascular injury. Recently we have shown that following radiation damage, transplanted hematopoietic stem cells (HSCs) give rise to endothelial cell outcomes at the clonal level. This raises the question of whether prospectively identifiable HSC progeny have endothelial cell activity. Using β-galactosidase and EGFP expression as donor markers, we show that lineage-restricted myeloid progenitors can give rise to endothelial cells. Transplanted common myeloid progenitors (CMPs) and granulocyte/macrophage progenitors (GMPs) gave rise to similar numbers of endothelial cells in liver portal vein branches. Donor-derived endothelial cells expressing functional endothelial markers including CD31, von Willebrand factor (vWF) and Tie-2 were clearly distinguished from mature hematopoietic cells by the absence of CD45 and F4/80 expression. Confocal microscopy showed the co-localization of donor and endothelial cell marker expression in individual cells so that endothelial cells could be discriminated from perivascular cells. To evaluate cell fusion as a potential mechanism for the emergence of endothelial cells of hematopoietic origin, HSCs transgenic for EGFP and Tie-2-Cre recombinase were transplanted into ROSA26 reporter (R26R) mice. Activation of β-galactosidase reporter gene expression, indicative of cell fusion with a donor-derived hematopoietic cell, could be detected in hepatocytes but not in endothelial cells. However, due to the tissue-specificity of the Tie-2 promoter, endothelial fusion products in which the donor nucleus had failed to activate the Tie-2-driven Cre recombinase would have also lacked detectable reporter gene expression. To ensure that the absence of β-galactosidase expression in the donor-derived endothelial cells was not due to insufficient nuclear reprogramming, HSCs transgenic for EGFP and R26R were transplanted into Tie-2-Cre recipients. Donor-derived endothelial cells were again negative for both β-galactosidase expression and activity. To determine whether radiation injury is required for the incorporation of donor cells into the endothelium, a parabiosis model was utilized. Both long-term donor-derived endothelial cell outcomes and multilineage hematopoiesis were detected in parabiotic mice. These results demonstrate that CMPs and further lineage-restricted GMPs are capable of differentiating into endothelial cells in the absence of cell fusion. Importantly, radiation injury is not required for endothelial cell engraftment, indicating that circulating progenitors contribute to angiogenesis during steady-state conditions.

In vivo myocardial gene transfer: optimization and evaluation of intracoronary gene delivery in vivo.

Clinical reports suggest that intracoronary delivery of adenoviruses encoding angiogenic growth factors, or their transactivators, has a therapeutic benefit. However there has not been a systematic assessment of the transfection efficiency of this technique in vivo. In rabbits we investigated the efficiency of myocardial gene transfer following intracoronary infusion of 1 x 10(-10) -1 x 10(12) p.f.u. of adenovirus in combination with interventions to enhance transfection. In five standard short axis sections, we were barely able to detect reporter gene expression following unmodified intracoronary infusion. Efficiency was not enhanced by the exclusion of blood and the increase of intracoronary dwell time through occlusive engagement of the left coronary ostium enabled by oxygenated perfluorocarbon emulsion as viral diluent. Of the interventions and pretreatments designed to increase vascular permeability, VEGF, calcium-free viral diluent and adenosine, only the latter tended to increase efficiency. However an intervention designed to increase the myocardial transcapillary gradient, by increasing venular pressure with pulmonary artery occlusion and arteriolar pressure with occlusion of the aorta above the coronary ostia, increased transfection efficiency by two orders of magnitude. Unfortunately the clinical utility of this technique may be limited by accompanying cardiac dilation and marked elevations in intracardiac pressure.

Rapid and sensitive detection of enhanced green fluorescent protein expression in paraffin sections by confocal laser scanning microscopy.

Various forms of green fluorescent protein (GFP) have become important reporters of gene transfer and expression after transfection or infection of cells in cell culture. Frequently, molecular biological assays (Northern blots, PCR) are applied to detect reporter gene expression in target organs. However, these methods are not suitable for evaluation of tissue- or cell-specific expression which would be of great interest especially in case of using tissue-specific promoters. Therefore, organs of transgenic mice with the enhanced green fluorescent protein (EGFP) gene under control of the cytomegalovirus (CMV) promoter were processed for histology by formaldehyde fixation and embedding in paraffin. Sections were deparaffinized, mounted and evaluated for fluorescence in a confocal laser scanning microscope. This method combines the advantages of direct exploitation of tissue sections without further staining procedures with evaluable tissue-, cell-, and even subcellular-specific distribution patterns of EGFP expression in tissues. Results obtained by direct evaluation of EGFP fluorescence in paraffin sections were confirmed by immunohistochemical staining with anti-EGFP. In the present report, we demonstrate that application of confocal microscopy on routinely processed histological preparations is very suitable for determining gene transfer efficiency and promotor activities.

CuZn-SOD promoter-driven expression in the Drosophila central nervous system☆

The aim of this study was to ascertain the status of CuZn superoxide dismutase (CuZn-SOD) expression in the central nervous system of Drosophila melanogaster. Immunoblot analysis of dissected tissue extracts revealed low levels of the CuZn-SOD protein in adult brains relative to other adult and larval tissues. To explore further this observation, three different reporter constructs containing different elements of the CuZn-SOD promoter domain were used for the generation of transgenic flies. A high level of reporter gene expression occurred during the second wave of neurogenesis (third instar and early pupal stages) in scattered, proliferating neuroblasts (NBs) and in proliferation centers of the optic lobe. In mature, postmitotic neurons, this expression was lower relative to other tissues. In adult flies, at all ages examined, there was little if any detectable reporter gene expression in cells of the central nervous system. These results suggest that one of the key components of the antioxidant defenses, CuZn-SOD, is quite low in postmitotic neural tissue, rendering it particularly susceptible to oxidative damage during aging.

Expression of the rat aldolase B gene: A liver-specific proximal promoter and an intronic activator

The nature and location of the cis-acting DNA sequences regulating expression of the rat aldolase B gene has been investigated. Two liver-specific DNAse I hypersensitive sites were detected, one located just upstream from the cap site, the second in the middle of the first, 4.8-kbp-long, intron. A fragment of 190 bp 5' to the cap site behaved as a tissue-specific but weak core promoter: it directed a detectable reporter gene expression in the Hep G2 cells and hepatocytes, but not in fibroblasts. The tissue-specific expression was stimulated at least 16 fold when constructs contained the entire first intron. The intronic activating sequences could be ascribed to an inner 2 kbp fragment in which the downstream liver-specific DNAse I hypersensitive site was located.