GFP Gene Research Articles

Imaging the invasion of rice roots by the bakanae agent Fusarium fujikuroi using a GFP-tagged isolate

Fusarium fujikuroi (teleomorph Gibberella fujikuroi) is the main seed-borne pathogen of rice, the causal agent of bakanae, a disease that in the last years has become of increasing economical concern in many Italian rice growing areas. A virulent F. fujikuroi isolate was tagged with the green fluorescent protein (gfp) gene using Agrobacterium tumefaciens mediated transformation, and the virulence of the GFP isolate has been confirmed. Little is known about the early interaction of the pathogen with its host, in this work fungal development during the F. fujikuroi/root interaction was analysed by Laser Scanning Confocal Microscopy (LSCM), by using the GFP isolate obtained. The infection of rice roots was investigated from 48 h to 8 days post-inoculation both in resistant and susceptible cultivars. Roots of resistant genotype seem to trigger a hypersensitive response at the infection site and LSCM analysis of root sections allowed the visualization of fungal growth within host tissues. Fungal growth occurred both in the resistant and the susceptible cultivar, even if it was less abundant in the resistant one. Expression analysis of Chitinase1, a gene involved in fungal pathogenesis, was investigated by qPCR on the F. fujikuroi infected rice roots. Chitinase1 expression increased greatly upon infection in the resistant cultivar Selenio.

Sensitive High-Throughput Assays for Tumour Burden Reveal the Response of a Drosophila melanogaster Model of Colorectal Cancer to Standard Chemotherapies.

Drosophila melanogaster (Drosophila) models of cancer are emerging as powerful tools to investigate the basic mechanisms underlying tumour progression and identify novel therapeutics. Rapid and inexpensive, it is possible to carry out genetic and drug screens at a far larger scale than in vertebrate organisms. Such whole-organism-based drug screens permits assessment of drug absorption and toxicity, reducing the possibility of false positives. Activating mutations in the Wnt and Ras signalling pathways are common in many epithelial cancers, and when driven in the adult Drosophila midgut, it induces aggressive intestinal tumour-like outgrowths that recapitulate many aspects of human colorectal cancer (CRC). Here we have taken a Drosophila CRC model in which tumourous cells are marked with both GFP and luciferase reporter genes, and developed novel high-throughput assays for quantifying tumour burden. Leveraging these assays, we find that the Drosophila CRC model responds rapidly to treatment with standard CRC-drugs, opening the door to future rapid genetic and drug screens.

Transformation of Long-Lived Albino Epipremnum aureum 'Golden Pothos' and Restoring Chloroplast Development.

Chloroplasts are organelles responsible for chlorophyll biosynthesis, photosynthesis, and biosynthesis of many metabolites, which are one of key targets for crop improvement. Elucidating and engineering genes involved in chloroplast development are important approaches for studying chloroplast functions as well as developing new crops. In this study, we report a long-lived albino mutant derived from a popular ornamental plant Epipremnum aureum ‘Golden Pothos’ which could be used as a model for analyzing the function of genes involved in chloroplast development and generating colorful plants. Albino mutant plants were isolated from regenerated populations of variegated ‘Golden Pothos’ whose albino phenotype was previously found to be due to impaired expression of EaZIP, encoding Mg-protoporphyrin IX monomethyl ester cyclase. Using petioles of the mutant plants as explants with a traceable sGFP gene, an efficient transformation system was developed. Expressing Arabidopsis CHL27 (a homolog of EaZIP) but not EaZIP in albino plants restored green color and chloroplast development. Interestingly, in addition to the occurrence of plants with solid green color, plants with variegated leaves and pale-yellow leaves were also obtained in the regenerated populations. Nevertheless, our study shows that these long-lived albino plants along with the established efficient transformation system could be used for creating colorful ornamental plants. This system could also potentially be used for investigating physiological processes associated with chlorophyll levels and chloroplast development as well as certain biological activities, which are difficult to achieve using green plants.

Programming gene expression in multicellular organisms for physiology modulation through engineered bacteria

A central goal of synthetic biology is to predictably and efficiently reprogram living systems to perform computations and carry out specific biological tasks. Although there have been many advances in the bio-computational design of living systems, these advances have mainly been applied to microorganisms or cell lines; programming animal physiology remains challenging for synthetic biology because of the system complexity. Here, we present a bacteria-animal symbiont system in which engineered bacteria recognize external signals and modulate animal gene expression, twitching phenotype, and fat metabolism through RNA interference toward gfp, sbp-1, and unc-22 gene in C. elegans. By using genetic circuits in bacteria to control these RNA expressions, we are able to program the physiology of the model animal Caenorhabditis elegans with logic gates. We anticipate that engineered bacteria can be used more extensively to program animal physiology for agricultural, therapeutic, and basic science applications.

Integration of [12]aneN3 and Acenaphtho[1,2-b]quinoxaline as non-viral gene vectors with two-photon property for enhanced DNA/siRNA delivery and bioimaging

Two-photon fluorescent Acenaphtho[1,2-b]quinoxaline (ANQ) and the hydrophilic di-(triazole-[12]aneN3) moieties were combined through an alkyl chain (ANQ-A-M) or a β-hairpin motif with two aromatic γ-amino acid residues (ANQ-H-M) to explore their capabilities for in vitro and in vivo gene delivery and tracing. ANQ-A-M and ANQ-H-M showed the same maximum absorption at 420 nm, and their fluorescent intensities around 650 nm were varied in different solvents and became poor in the protic solvents. Gel electrophoresis assays indicated that both compounds completely retarded the migration of pDNA at 20 μM in the presence of DOPE. However, the DNA condensation with ANQ-H-M was not reversible, and the particle size of the corresponding complexes were larger indicated from the SEM and DLS measurements. In vitro transfections indicated ANQ-A-M/DOPE achieved Luciferase and GFP expressions were to be 7.9- and 5.7-fold of those by Lipo2000 in A549 cells respectively. However, ANQ-H-M showed very poor transfection efficiency in Luciferase expression. With the help of single/two-photon fluorescence imaging it clearly demonstrated that the successful transfection of ANQ-A-M was attributed to its cellular uptake, apparent lysosomal escape, and reversible release of DNA; and the poor transfection of ANQ-H-M was resulted from the aggregation of the DNA complexes which prevented them from the cellular uptake, and also the strong binding ability which is not easy to release DNA. ANQ-A-M/DOPE also exhibited robust gene silencing (83% knockdown of Luciferase) and GFP expression (2.47-fold higher) efficiency compared with Lipo2000 in A549 and zebrafish, respectively. The work demonstrated that the linkage structure between fluorescent and di(triazole-[12]aneN3) played the important role for their gene delivery performance, and that ANQ-A-M represents a vector with the strong transfection efficiency in vitro and in vivo as well as the efficient real time bioimaging properties, which is potential for the development in biomedical research.

CRISPR-mediated base editing in mice using cytosine deaminase base editor 4

Background: Many human genetic diseases arise from point mutations. These genetic diseases can theoretically be corrected through gene therapy. However, gene therapy in clinical application is still far from mature. Nearly half of the pathogenic single-nucleotide polymorphisms (SNPs) are caused by G:C>A:T or T:A>C:G base changes and the ideal approaches to correct these mutations are base editing. These CRISPR-Cas9-mediated base editing does not leave any footprint in genome and does not require donor DNA sequences for homologous recombination. These base editing methods have been successfully applied to cultured mammalian cells with high precision and efficiency, but BE4 has not been confirmed in mice. Animal models are important for dissecting pathogenic mechanism of human genetic diseases and testing of base correction efficacy in vivo. Cytidine base editor BE4 is a newly developed version of cytidine base editing system that converts cytidine (C) to uridine (U). Results: In this study, BE4 system was tested in cells to inactivate GFP gene and in mice to introduce single-base substitution that would lead to a stop codon in tyrosinase gene. High percentage albino coat-colored mice were obtained from black coat-colored donor zygotes after pronuclei microinjection. Sequencing results showed that expected base changes were obtained with high precision and efficiency (56.25%). There are no off-targeting events identified in predicted potential off-target sites. Conclusions: Results confirm BE4 system can work in vivo with high precision and efficacy, and has great potentials in clinic to repair human genetic mutations. Normal 0 false false false EN-US X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:Tabla normal; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin-top:0cm; mso-para-margin-right:0cm; mso-para-margin-bottom:8.0pt; mso-para-margin-left:0cm; line-height:107%; mso-pagination:widow-orphan; font-size:11.0pt; font-family:Calibri,sans-serif; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:Times New Roman; mso-bidi-theme-font:minor-bidi;}

Bi-functionalized aminoguanidine-PEGylated periodic mesoporous organosilica nanoparticles: a promising nanocarrier for delivery of Cas9-sgRNA ribonucleoproteine

BackgroundThere is a great interest in the efficient intracellular delivery of Cas9-sgRNA ribonucleoprotein complex (RNP) and its possible applications for in vivo CRISPR-based gene editing. In this study, a nanoporous mediated gene-editing approach has been successfully performed using a bi-functionalized aminoguanidine-PEGylated periodic mesoporous organosilica (PMO) nanoparticles (RNP@AGu@PEG1500-PMO) as a potent and biocompatible nanocarrier for RNP delivery.ResultsThe bi-functionalized MSN-based nanomaterials have been fully characterized using electron microscopy (TEM and SEM), nitrogen adsorption measurements, thermogravimetric analysis (TGA), X-ray powder diffraction (XRD), Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy (ATR-FTIR), and dynamic light scattering (DLS). The results confirm that AGu@PEG1500-PMO can be applied for gene-editing with an efficiency of about 40% as measured by GFP gene knockdown of HT1080-GFP cells with no notable change in the morphology of the cells.ConclusionsDue to the high stability and biocompatibility, simple synthesis, and cost-effectiveness, the developed bi-functionalized PMO-based nano-network introduces a tailored nanocarrier that has remarkable potential as a promising trajectory for biomedical and RNP delivery applications.

Agrobacterium tumefaciens Mediated Transformation of Symbiodinium spp.

Symbiodinium spp conducts symbiosis mutualism within a wide phyletic range of marine invertebrate hosts including corals and anemones. The present study investigates the transformation of foreign genes into the free living cultured Symbiodinium spp by co-cultured Symbiodinium cells with A. tumefaciens. Ti-plasmidbased binary vector harboring the GUS and GFP genes were transformed into Symbiodinium cells by co-cultivation. GUS histochemical assay was monitored in Symbiodinium cells under light microscopy. Putative GFP in transformed Symbiodinium cells was detected by immunoassaying with antibodies against GFP protein. These results suggest that A. tumefaciens could provide efficient tools for gene transformation of Symbiodinium cells.

Creation of an inducible vector system based on the rhizobia nodA gene promoter

BACKGROUND: The possibility of changing the properties of rhizobial bacteria by giving them the ability to regulate the expression of additionally introduced genes into them is an urgent task both for fundamental science and for applied agrobiology, since this will make it possible to obtain microsymbionts with desired properties. An expression construct using the rhizobia regulatory system was created in this work. The rhizobia nodD gene encodes a regulatory protein that, in the presence of plant inducers, flavonoids, activates the transcription of nod-genes involved in the early stages of the formation of legume-rhizobium symbiosis.
 MATERIALS AND METHODS: A vector construct containing the nodD gene from Rhizobium leguminosarum bv. trifoli under the regulation of its own promoter and the gfp gene under the regulation of the nodA gene promoter from the same rhizobia was obtained. Neorhizobium galegae CIAM 0702 were transformed with the vector construct.
 RESULTS: It has been shown that in recombinant strains synthetic flavonoids are capable of inducing expression of gfp gene to varying degrees.
 CONCLUSION: In the future, the results can be used to obtain rhizosphere microorganisms with a controlled synthesis of growth-stimulating and protective substances.

Marker counter-selection via CRISPR/Cas9 co-targeting for efficient generation of genome edited avian cell lines and germ cells

Efficient isolation of genetically modified cells that are phenotypically indistinguishable from the unmodified cells remains a major technical barrier for the broader utilization of CRISPR/Cas9. Here, we report a novel enrichment approach to select the genome engineered cells by co-targeting a genomically integrated GFP gene along with the endogenous gene of interest (GOI). Using this co-targeting approach, multiple genomic loci were successfully targeted in chicken (DF1) and quail (CEC-32) fibroblast cell lines by transient transfection of Cas9 and guide RNAs (gRNAs). Clonal isolation of co-targeted DF1 cells showed 75% of cell clones had deletion of GFP and biallelic deletion of the GOI. To assess the utility of this approach to generate genome modified animals, we tested it on chicken primordial germ cells (PGCs) expressing GFP by co-targeting with gRNAs against GFP and endogenous ovomucoid (OVM) gene. PGCs enriched for loss of GFP and confirmed for OVM deletion, derived by co-targeting, were injected into Hamburger and Hamilton stage 14–15 chicken embryos, and their ability to migrate to the genital ridge was confirmed. This simple, efficient enrichment approach could easily be applied to the creation of knock-out or edited cell lines or animals.

Potato virus X-mediated constitutive expression of Plutella xylostella PxSDF2L1 gene in Nicotiana benthamiana confers resistance to Phytophthora parasitica var. nicotianae

BackgroundThe Plutella xylostella PxSDF2L1 gene was previously reported to enhance insect resistance to pathogen at high basal transcription rate. PxSDF2L1 shows similitude with the stromal cell-derived factor 2 (SDF2), an ER stress-induced chaperon protein that is highly conserved throughout animals and plants. The precise biological function of SDF2 is not clear, but its expression is required for innate immunity in plants. Here, we investigate whether a continuous expression of PxSDF2L1 in Nicotiana benthamiana can similarly confer resistance to plant pathogen, particularly, the black shank Phytophthora parasitica var. nicotianae.ResultsThe N. benthamiana plants were inoculated with agrobacteria transformed with a PVX-based binary vector carrying the PxSDF2L1 gene; similar agroinoculation experiments with a PVX vector carrying the GFP gene were used for controls. In pot trials, agroinfected N. benthamiana plants constitutively expressing PxSDF2L1 showed a significant reduction of stem disease symptoms caused by the inoculation with P. parasitica, compared with controls.ConclusionsWe confirm a role of PxSDF2L1 in resistance to black shank, with a potential application to engineering active resistance against this oomycete in the commercial N. tabacum species and propose its evaluation in other crop families and plant pathogens.

Intravenous infusion of mesenchymal stem cells delays disease progression in the SOD1G93A transgenic amyotrophic lateral sclerosis rat model

ALS is a devastating neurodegenerative disease with few curative strategies. Both sporadic and familial ALS display common clinical features that show progressive paralysis. The pathogenesis remains unclear, but disruption of the blood-spinal cord barrier (BSCB) may contribute to the degeneration of motor neurons. Thus, restoration of the disrupted BSCB and neuroprotection for degenerating motor neurons could be therapeutic targets. We tested the hypothesis that an intravenous infusion of MSCs would delay disease progression through the preservation of BSCB function and increased expression of a neurotrophic factor, neurturin, in SOD1G93A ALS rats. When the open-field locomotor function was under 16 on the Basso, Beattie, and Bresnahan (BBB) scoring scale, the rats were randomized into two groups; one received an intravenous infusion of MSCs, while the other received vehicle alone. Locomotor function was recorded using BBB scoring and rotarod testing. Histological analyses, quantitative reverse transcription-polymerase chain reaction (qRT-PCR), were performed. The MSC group exhibited reduced deterioration of locomotor activity compared to the vehicle group, which displayed progressive deterioration of hind limb function. We observed the protection of motor neuron loss and preservation of microvasculature using Evans blue leakage and immunohistochemical analyses in the MSC group. Confocal microscopy revealed infused green fluorescent protein+ (GFP+) MSCs in the spinal cord, and the GFP gene was detected by nested PCR. Neurturin expression levels were significantly higher in the MSC group. Thus, restoration of the BSCB and the protection of motor neurons might be contributing mechanisms to delay disease progression in SOD1G93A ALS rats.

Some Special Aspects of Liver Repair after Resection and Administration of Multipotent Stromal Cells in Experiment

Changes in rat liver after resection and injection of autologous multipotent mesenchymal stromal cells of bone marrow origin (MSCs) transfected with the GFP gene and cell membranes stained with red-fluorescent lipophilic membrane dye were studied by light microscopy. It was found that after the introduction of MSCs into the damaged liver, their differentiation into any cells was not found. However, under the conditions of MSCs use, the number of neutrophils in the parenchyma normalizes earlier, and necrosis and hemorrhages disappear more quickly. It was concluded that the use of MSCs at liver resection for the rapid restoration of an organ is inappropriate, since the injected cells in vivo do not differentiate either into hepatocytes, into epithelial cells of bile capillaries, into endotheliocytes and pericytes of the vascular membranes, into fibroblasts of the scar or other connective tissue structures, or into any other cells present in the liver.

Evaluation of Direct and Cell-Mediated Lactoferrin Gene Therapy for the Maxillofacial Area Abscesses in Rats.

Resistance to antibacterial therapy requires the discovery of new methods for the treatment of infectious diseases. Lactoferrin (LTF) is a well-known naïve first-line defense protein. In the present study, we suggested the use of an adenoviral vector (Ad5) carrying the human gene encoding LTF for direct and cell-mediated gene therapy of maxillofacial area phlegmon in rats. Abscesses were developed by injection of the purulent peritoneal exudate in the molar region of the medial surface of the mandible. At 3–4 days after phlegmon maturation, all rats received ceftriaxone and afterward were subcutaneously injected around the phlegmon with: (1) Ad5 carrying reporter gfp gene encoding green fluorescent protein (Ad5-GFP control group), (2) Ad5 carrying LTF gene (Ad5-LTF group), (3) human umbilical cord blood mononuclear cells (UCBC) transduced with Ad5-GFP (UCBC + Ad5-GFP group), and (4) UCBC transduced with Ad5-LTF (UCBC + Ad5-LTF group). Control rats developed symptoms considered to be related to systemic inflammation and were euthanized at 4–5 days from the beginning of the treatment. Rats from therapeutic groups demonstrated wound healing and recovery from the fifth to seventh day based on the type of therapy. Histological investigation of cervical lymph nodes revealed purulent lymphadenitis in control rats and activated lymphatic tissue in rats from the UCBC + Ad5-LTF group. Our results propose that both approaches of LTF gene delivery are efficient for maxillofacial area phlegmon recovery in rats. However, earlier wound healing and better outcomes in cervical lymph node remodeling in the UCBC + Ad5-LTF group, as well as the lack of direct exposure of the viral vector to the organism, which may cause toxic and immunogenic effects, suggest the benefit of cell-mediated gene therapy.

MicroRNA-378 regulates adipogenic differentiation in bovine intramuscular preadipocytes by targeting CaMKK2

ABSTRACT Intramuscular fat, as one of the most important palatability attribute of beef carcase, is the primary determinant of beef quality. The research of adipogenesis mechanism would provide new insight into intramuscular fatty deposition. Here, the role of microRNA-378 was investigated during bovine adipogenic differentiation. It was revealed that miR-378 expression exists variably in bovine major tissue and organs by RT-qPCR. It was predicted that miR-378 targets CaMKK2, as an AMPKα kinase, by DIANA Tools. For better research, primary preadipocytes with stable transfection for up-/down-regulated expression of miR-378 were constructed by lentiviral vectors with GFP gene. The analyses of qPCR showed that PPARγ and adiponectin mRNA levels increased, but C/EBPβ, pref-1 and CaMKK2 mRNA levels decreased during adipogenic differentiation. When miR-378 was overexpressed, preadipocytes proliferation became slower, there are more cellular lipid droplets, and PPARγ and C/EBPβ mRNA levels were higher, but pref-1, adiponectin and CaMKK2 were lower than control groups. Luciferase assay and western blot analysis validated that miR-378 binds the nucleotide sites of the 3′- untranslated region of CaMKK2, which inhibits the mRNA and protein expression of CaMKK2. These findings suggest that miR-378 promotes adipogenic differentiation in bovine intramuscular preadipocytes by targeting CaMKK2 via AMPK signalling pathway.

Development of mesothelin-specific CAR NK-92 cells for the treatment of gastric cancer.

Background: The application of chimeric antigen receptor (CAR) NK cells in solid tumors is hindered by lack of tumor-specific targets and inefficient CAR NK cell efficacy. It has been reported that mesothelin (MSLN) may be an ideal immunotherapy target for gastric cancer. However, the feasibility of using anti-MSLN CAR NK cells to treat gastric cancer remains to be studied.Methods: MSLN expression in primary human gastric cancer, normal tissues and cell lines were detected. MSLN and CD19 targeted CAR NK-92 (MSLN- and CD19-CAR NK) cells were constructed, purified and verified. N87, MKN-28, AGS and Huh-7 cells expressing the GFP and luciferase genes were transduced. Cell- and patient-derived xenograft (PDX) were established via NSG mice. The ability of MSLN-CAR NK cells to kill MSLN-positive gastric cancer cells were evaluated in vitro and in vivo.Results: MSLN-CAR NK cells can specifically kill MSLN-positive gastric cancer cells (N87, MKN-28 and AGS), rather than MSLN negative cell (Huh-7), in vitro. Moreover, compared with parental NK-92 cells and CD19-CAR NK cells, stronger cytokine secretions were secreted in MSLN-CAR NK cells cocultured with N87, MKN-28 and AGS. Furthermore, MSLN-CAR NK cells can effectively eliminate gastric cancer cells in both subcutaneous and intraperitoneal tumor models. They could also significantly prolong the survival of intraperitoneally tumor-bearing mice. More importantly, the potent antitumor effect and considerable NK cell infiltration were observed in the patient-derived xenograft treated with MSLN-CAR NK cells, which further warranted the therapeutic effects of MSLN-CAR NK cells to treat gastric cancer.Conclusion: These results demonstrate that MSLN-CAR NK cells possess strong antitumor activity and represent a promising therapeutic approach to gastric cancer.

A Facile Method to Engineer Mutant Kras Alleles in an Isogenic Cell Background.

Oncogenic KRAS mutations are common in colorectal cancer (CRC), found in ~50% of tumors, and are associated with poor prognosis and resistance to therapy. There is substantial diversity of KRAS mutations observed in CRC. Importantly, emerging clinical and experimental analysis of relatively common KRAS mutations at amino acids G12, G13, A146, and Q61 suggest that each mutation differently influences the clinical properties of a disease and response to therapy. Although clinical evidence suggests biological differences between mutant KRAS alleles, these differences and the mechanisms underlying them are not well understood, and further exploration of allele-specific differences may provide evidence for individualized therapeutics. One approach to study allelic variation involves the use of isogenic cell lines that express different endogenous KRAS mutants. Here we developed an assay using fluorescent co-selection for CRISPR-driven gene editing to generate various Kras mutations in an isogenic murine colon epithelial cell line background. This assay involves generation of a cell line stably expressing Cas9 linked to BFP and simultaneous introduction of single-guide RNAs (sgRNAs) to two different gene loci resulting in double-editing events. Single-stranded donor oligonucleotides are introduced for a GFP gene and a Kras mutant allele of our choice as templates for homologous recombination (HDR). Cells that successfully undergo HDR are GFP-positive and have a higher probability of containing the desired Kras mutation. Therefore, selection for GFP-positive cells allows us to identify those with phenotypically silent Kras edits. Ultimately, this method allows us to toggle between different mutant alleles and preserve the wild-type allele while maintaining an isogenic background.

The Escherichia coli azoR gene promoter: A new sensing element for microbial biodetection of trace explosives

Abstract DNT (2,4-dinitrotoluene), a volatile impurity in military grade TNT-based explosives, is a potential tracer for the detection of buried landmines and other explosive devices. We have previously described DNT bioreporter strains, based on a fusion between the Escherichia coli yqjF gene promoter to either a bacterial luxCDABE luminescence gene cassette or to the green fluorescent protein gene gfp. We now present the azoR gene promoter, demonstrated to be strongly induced by DNT, as an alternative sensing element for this compound. An E. coli bioreporter strain harboring a plasmid-borne fusion of the azoR promoter region to Photorhabdus luminescens luxCDABE displayed stronger bioluminescent responses to DNT than the yqjF-based bioreporter. The azoR promoter was induced not directly by DNT but by its degradation products; the most potent inducer was 2,4,5-trihydroxytoluene (THT). This reaction was positively regulated by YhaJ, a member of the LysR family of transcriptional regulators, and was significantly amplified in a yhaK mutant. We also show that DNT degradation in E. coli involves glutathione, which plays a vital role in azoR gene promoter activation.

The 2′-caged-tethered-siRNA shows light-dependent temporal controlled RNAi activity for GFP gene into HEK293T cells

Small interfering RNA (siRNA) exhibits gene-specific RNAi activity by the formation of RISC complex with mRNA of gene. The structural modification of siRNA at appropriate positions affects the structure of RISC complex and then RNAi activity. The modified siRNA are mostly prepared from the incorporation of sugar ring modified, and nucleobase modified RNA nucleotides. It is learned that the introduction of the sterically hindered nucleoside at the specific position of siRNA, severely affects siRNA-RISC complex formation. This report describes the syntheses of bulkier siRNA from 2′-caged-tethered-siRNAs, containing bulkier photolabile protecting group (o-nitrobenzyl) at 2′-position of ribose nucleoside. Importantly, these 2′-caged-siRNAs exhibit the light-dependent RNA interference (RNAi) activity into HEK293T cells for the GFP gene expression. The 2′-caged-siRNAs are synthesized by caging the sense and antisense strand of siRNA. The biochemical evaluations of these caged-siRNAs show that antisense-strand caged-siRNAs decrease RNAi activity temporarily in dark while enhancing RNAi activity, almost like control, after exposure withUV- light. However, 2′-caged sense strand siRNA increase RNAi activity temporarily while decreasing RNAi activity after exposure with light. These caged-siRNAs are also stable in the serum (fetal bovine serum) as like native siRNA. Hence these results strongly support that 2′-caged-tethered-siRNAs are promising analogues to control RNAi activity by UV-light.

Development of a massively parallel reporter assay to identify functional regulatory variants in the PICALM Alzheimer disease associated locus

AbstractBackgroundMost significant genome‐wide association markers for Alzheimer disease (AD) are in noncoding portions of the genome making assigning a corresponding gene involved in risk challenging. Coding variants identified in some these GWAS loci thus far do not explain the proportion of risk attributable to the associated markers, indicating a contribution of noncoding variants to AD. We set out to develop a high throughput method investigating the functional role of noncoding variants associated with AD using the PICALM locus on chromosome 14:85,600,000‐85,860,000, which has been associated with AD across multiple ancestries, as a proof of concept region.MethodWe used the gnomAD database of over 15,000 whole genomes to identify all variants located in the PICALM locus across a wide variety of ancestral populations (non‐Hispanic whites, Hispanics, African Americans, and Asians). We developed a pipeline for epigenomic annotation from available databases and the subsequent design of oligos carrying wild‐type and variant alleles for each variant. For unbiased analyses of functional potential to regulate enhancer activity of each of the alleles we established a large vector library for massively parallel reporter assays (MPRA) in AD relevant cell lines.ResultWe identified >23,000 variants for analysis in the 260 kb PICALM locus. More than 4,000 of those had prior evidence for location in transcription factor binding sites, putative promotors/enhancers and eQTLs. 180 bp oligos centered around the variant alleles were synthesized by TWIST Biosciences. 20 bp random barcodes were added to each of the oligos through PCR amplification with a barcoded primer library. Sequencing of the resulting vector library indicated highly efficient incorporation with 95% unique barcodes present in the library. Reporter gene GFP and a minimal promoter were subsequently cloned in between the oligo and the barcode to establish the final MPRA library for transformation in cell lines.ConclusionHere we established a computational framework for in‐silico epigenomic annotation and oligo design for subsequent high‐throughput analyses and developed an efficient protocol to create a final MPRA library with highly unique barcodes at fragment incorporation. Cell line transformations and transcriptome analyses of the PICALM library to identify regulatory variants are ongoing.