Beta-galactosidase Reporter Plasmid Research Articles

Abstract A21: RISCI: A high-throughput screening platform for investigating apoptosis and toxicity signaling.

Abstract Apoptosis signaling is fundamental to disease progression and therapeutic success. Our group have developed a high-throughput genetic screening platform, named RISCI, with a daily throughput of 1,536 unique clones. By combining the wealth of genomics data with RISCI results, we attempt to identify new regulators of apoptosis signaling networks and hope that this approach offers insights into the regulation of compound efficacy and disease progression. Nanotechnology has been garnering immense interest, with potential applications for drug delivery and cancer treatments. However, the implementation of this new technology is hampered by the current limited knowledge. Some of these nanoparticles, in particular those at the lower size range, are cytotoxic due to increased surface area to volume ratio that enhances catalysis activity. The RISCI screening platform comprises a pair of custom-made robots handling DNA isolation and transfection, respectively. Ultrapure plasmid DNA is isolated and co-transfected with a beta-galactosidase reporter plasmid using an in-house developed protocol based on polyethylenimine polymer. The genes in our human cDNA library (NITE), a collection of approximately 30,000 full-length and sequenced genes, are systematically screened for causing membrane permeability. The integrity of the plasma membrane in apoptotic cells is damaged, allowing for the entry and turnover of the substrate to generate a colorimetric signal. Lysis with Triton after an initial measurement facilitates the assessment of transfection efficiency and readout normalization. Apoptosis-inducing candidates are then identified from a positive signal ratio. The use of silica nanoparticles extends the capacity of the RISCI screen to discover modulators of its toxicity. Initial results suggest that one of our apoptosis inhibitors is able to inhibit the cell death caused by these nanoparticles. Such an effect can also be assayed using the screen assay system, allowing for the identification of additional apoptosis inhibitors and sensitizers. While inhibitors are selected using a low signal ratio upon treatment with cytotoxic agents such as silica nanoparticles, genes sensitizing the cells to apoptosis upon treatment are highlighted by an increased signal ratio. A previous screen using a mouse cDNA library assayed in excess of 100,000 redundant clones and yielded 74 verified apoptosis inducers, including Orctl3, which was shown to have tumor-specific activity. The NITE library has significantly less redundancy, and current progress of approximately 4,000 clones identified 48 inducer candidates. The current discovery rate of 1.2% is much higher than previous attempts due to the reduced redundancy. These candidates will be subjected to further apoptosis assays such as Roche Nucleosome ELISA kit and PARP cleavage. Upon completion of the screen, 350–500 apoptosis inducing candidates are expected to be identified. Data are also being analyzed for inhibitors and sensitizers identification. The list of apoptosis inducers and inhibitors, when combined with the available microarrays and “omics” data, will allow us to identify new drug targets if the specific gene is implicated in a degenerative disease or cancer. The data from the nanoparticles screen can also identify new biomarkers that are specific to nanoparticle toxicity as part of a risk assessment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A21.

Correct evaluation of reporter assays in different cell lines by direct determination of the introduced plasmid amount

Transfection efficiency in reporter gene assays is usually determined by cotransfection of a reference reporter gene under the control of a constitutively active strong promoter and determination of the reference enzyme activity. The SV40 promoter-driven beta-galactosidase reporter plasmid is frequently used as the reference reporter plasmid. Here we show that the beta-galactosidase expression in different cell lines does not correctly reflect the amount of plasmid taken up by cells and thus is not an accurate measure of transfection efficiency. The direct determination of introduced plasmid concentration in lysates of transfected cells is suitable for monitoring the transfection efficiency in reporter gene assays even if different cell lines are compared.

Improved lipid-mediated gene transfer in C6 glioma cells and primary glial cells using FuGene™

Gene therapy is a potent method to counteract neurodegeneration by introducing genetic information encoding neuroprotective factors. In this study cationic lipids were used to transfer DNA into C6 glioma cells and primary glial cells. When comparing the novel compound FuGene with other commercially-available lipids, it was found that FuGene markedly enhanced gene transfer of a beta-galactosidase reporter plasmid into C6 glioma cells. FuGene had several advantages compared to other lipids, such as a very low toxicity and the capability of transfection under serum conditions. When optimizing, a DNA–lipid ratio of 150 ng DNA/1 μl FuGene and a concentration of 3 μl FuGene/1 ml medium was found to be optimal. The incubation time peaked after 8 h and the expression time reached an optimum between 2 and 6 days. When cells were transfected on 3 consecutive days for 6 h each (‘boosting’), the transfection efficiency was markedly enhanced in primary glial cells. When using endotoxin-free DNA the transfection efficiency could be enhanced up to 3 times. The optimal transfection efficiency in C6 glioma cells and in primary glial cells was found to be 16.3±0.3% and 5.1±0.37% of total cells, respectively. In conclusion this study shows that the novel compound FuGene has a very high potential to transfer DNA into cells of glial origin, and it might be an interesting canditate for ex vivo and in vivo gene therapeutic approaches.

Gene transfer into rat heart-derived endothelial cells.

Progressive graft arteriosclerosis is responsible for the majority of late deaths in cardiac transplant recipients. Despite many investigations, the pathogenesis of this disease remains undetermined and its control inadequate. A somatic gene transfer during the cold ischemic time and thus before transplantation might be a new therapeutic tool. This approach allows a long incubation time of the DNA and a safe transfer with liposomes and transferrin with less adverse effects for the organ recipient. The target cells (microvascular endothelial cells (MVECs)) for this gene transfer were isolated from rat hearts by perfusion with collagenase via an aortic cannulae. The cells were purified by changing the medium 30 min after subcultivation in order to remove fibroblasts and smooth muscle cells. The endothelial cells (ECs) were identified by typical morphology and the uptake of Dil-Ac-LDL. The gene transfer was carried out with a beta-galactosidase reporter plasmid (pCMVbeta), cationic liposomes (Lipofectin), and transferrin. Different transfection solutions were prepared with or without serum, and with different plasmid-liposome ratios and transferrin concentrations. The transfer rate was monitored with a semiquantitative orthonitrophenyl-beta/-D-galactoside (ONPG) assay and histologically by X-Gal staining. The cytotoxicity of this procedure was determined with a colorimetric ELISA with Alamar blue. The cardioplegic property of the transfection solution was tested in a Langendorff perfusion system monitoring the coronary blood flow over time after a cold ischemic time of 4 h. The maximal gene expression could be detected after transfection with 4 microl Lipofectin, 2 microg pCMVbeta, and 16 microg transferrin/200 microl transfection solution. Under these conditions 60% of the cells showed a blue staining with X-Gal. Only 20% of the cells died during transfection. The lowest cytotoxicity during cold ischemic time for ECs was assessed with normal cell culture medium and the Buckberg solution. The best coronary flow rates after 4 h cold ischemia of the heart were measured for cardioplegia with St. Thomas and Buckberg solutions. In summary, the best transfection solution with a good cardioplegic property was the Buckberg solution. Finally, the results of this study show that an effective DNA delivery with a low toxicity into ECs is possible with a combination of liposomes and transferrin. This method might be useful for a safe and effective gene transfer into solid organs during the cold ischemic time and thus a therapeutic tool for chronic rejection.

Transcriptional regulation of the tissue factor gene by progestins in human endometrial stromal cells.

Decidualization of estradiol (E2)-primed human endometrial cells (HESCs) by progesterone is associated with elevated levels of tissue factor (TF), the primary initiator of hemostasis. Similarly, in cultured human HESCs, the synthetic progestin, medroxyprogesterone acetate (MPA), enhances TF protein and messenger ribonucleic acid (mRNA) levels. Although ineffective alone, E2 potentiates this progestin enhancement of TF expression by HESCs. The current study examines mechanisms underlying MPA enhancement of TF mRNA expression in HESCs. In the presence of the transcription-blocking agent dichlororibofuranosylbenzimidazole, no significant differences were noted in the half-lives of TF mRNA isolated from HESCs treated with E2 alone or with E2 plus MPA. This indicates that MPA-enhanced TF mRNA levels do not reflect changes in the stability of the TF message. To test the effect of progestin on TF promoter activity and to ascertain the mechanism of promoter regulation, primary or first passaged HESCs were transfected with TF promoter constructs spanning the regions -2106 to +121 (TFp(-2106)), -278 to +121 (TFp(-278)), and -111 to +14 (TFp(-111)) bp upstream of the transcription start site. MPA was found to enhance TF transcription by 20-fold in HESCs transfected with TFp(-2106) after correcting for transfection efficiencies with a beta-galactosidase reporter plasmid. Interestingly, levels of E2- plus MPA-stimulated transcription were significantly increased using TFp(-278) compared to TFp(-2106), suggesting that the region between -2106 and -278 bp may contain an inhibitory element. In addition, rates of MPA-stimulated transcription using TFp(-111) were significantly reduced compared to values obtained using TFp(-2106) and were even further reduced compared to values obtained using TFp(-278). This suggests that regulatory elements in the -111 bp region of the TF promoter are necessary for progestin-mediated regulation of the TF gene in HESCs, but are not sufficient to account for maximal rates of TF gene transcription. Our results also demonstrated that induction of steady state TF mRNA by MPA was abolished by treating cells with E2 plus MPA in conjunction with the protein synthesis inhibitor cycloheximide. In light of the absence of a complete progesterone or estrogen response element in the published 5'-sequence of the TF promoter, our results suggest that progestin-enhanced transcription of TF mRNA in stromal cells may be mediated by an uncharacterized protein intermediate(s).

Histone H2A Significantly Enhances In Vitro DNA Transfection

Gene transfer is a potential treatment modality of genetic disease. Efficient, practical methods of DNA transfection are currently under investigation. A beta-galactosidase reporter plasmid interacted electrostatically with histones, poly-L-Lys, poly-L-Arg, and a combination of poly-L-Lys and poly-L-Arg. This complex was then used to transfect COS-7 cells. beta-galactosidase activity was quantified and used to compare the efficiency of gene transfection in vitro. A comparison was also made of DNA transfection with the most active histone subclass, i.e., histone H2A, in the absence and presence of an anionic liposome. There was a marked increase in DNA transfection in the presence of histone H2A when compared with the control, whereas each of the other histones and polycations showed little, if any, effect. The extent of activation depends strongly on the DNA/histone ratio and is also a function of the molarity of the final Tris-acetate, pH 8, solution. The anionic liposomes used demonstrated an inhibitory effect. Histone H2A significantly enhances in vitro DNA transfection whereas other histones and anionic liposomes do not. A study of the difference between histone H2A and other histone subclasses may serve to clarify some of the mechanisms and the essential components of efficient gene delivery.

Biochemical and functional analysis of an adenovirus-based ligand complex for gene transfer.

Ligand-mediated approaches to gene transfer offer an alternative to viral vectors for both in vivo and in vitro applications. Although a significant percentage of the plasmid-based DNA complex is lost to lysosomal degradation following receptor-mediated endocytosis, simultaneous infection with adenovirus has been shown to increase the level of transgene expression [Curiel, Agarwal, Wagner and Cotten (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 8850-8854; Wagner, Zatloukal, Cotten, Kirlappos, Mechtler, Curiel and Birnstiel (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 6099-6103]. In this study we describe an adenovirus-based ligand complex where the plasmid DNA, polycation-ligand conjugate and adenovirus are contained within a single particle structure. At the core of the transfection particle is a replication-defective recombinant adenovirus encoding a cDNA minigene for human placenta alkaline phosphatase that was chemically modified with poly(L-lysine) (Ad-pLys). Electron microscopy of an adenovirus-based ligand complex formed by successively adding plasmid DNA and an asialo-orosomucoid-poly(L-lysine) conjugate to Ad-pLys revealed structures that appeared as intact viral particles coated with a dense biomolecular layer. Adenovirus-based ligand complexes containing either a luciferase or beta-galactosidase reporter plasmid were shown to efficiently deliver the plasmid transgene to cells that express the hepatic asialoglycoprotein receptor. Furthermore, the poly(L-lysine) modification greatly reduced the infectivity potential of the virus without causing a concomitant loss of augmented gene transfer. As an alternative to infectious virions, incomplete products of viral assembly were also considered as a source for endosomalytic activity. However, these defective virions were unable to significantly enhance plasmid transgene delivery.