RNA-seq Analysis
Altuna Akalin in Computational Genomics with R, 2020
Quantification of how much gene expression levels deviate from a baseline gives clues about which genes are actually important for, for instance, disease outcome or cell/tissue identity. The methods of detecting and quantifying gene expression have evolved from low-throughput methods such as the usage of a reporter gene with a fluorescent protein product to find out if a single gene is expressed at all, to high-throughput methods such as massively parallel RNA-sequencing that can profile -at single-nucleotide resolution- the abundance of tens of thousands of distinct transcripts encoded in the largest eukaryotic genomes.
Imaging of Cell Trafficking and Cell Tissue Homing
George C. Kagadis, Nancy L. Ford, Dimitrios N. Karnabatidis, George K. Loudos in Handbook of Small Animal Imaging, 2018
Indirect labeling procedures rely on the detection of a reporter gene. For these applications, a reporter gene is introduced into the cell, which is transformed into a nonnative enzyme, receptor, or fluorescent or bioluminescent protein that accumulates and can be detected (Weissleder et al. 2000; Ponomarev et al. 2004). If the reporter gene expression is stable, the cells can be monitored over their entire lifetime and information about cell proliferation, activation, or death can be extracted. Although the cell label is passed on to the next generation without dilution, the reporter gene can be silenced, which would suppress the signal (Krishnan et al. 2006). Potential disadvantages include costs, cellular dysfunction or death, immunogenicity of gene products, and potential risk of uncontrolled growth and malignancy. As a result, indirect labeling is possible only in preclinical settings, as these aspects preclude clinical application in patients at this time.
Delivery of Genes Through the Lung Circulation
Kenneth L. Brigham in Gene Therapy for Diseases of the Lung, 2020
Gene delivery to the pulmonary circulation is feasible. Using present technology, isolation of the lung circulation with either a catheter-based or a surgical approach yields the highest expression. Both lipid and adenoviral vectors yield lung expression, though adenovirus produces higher efficiency. While systemic delivery utilizing lipid:DNA complexes appears to result primarily in endothelial cell expression, the higher levels of expression achieved with directed approaches result in expression in a variety of other lung cells, including alveolar epithelial cells. Of note, little expression in vascular smooth muscle cells has been detected in any study to date. The present studies still represent early stages of development of this technology. While a variety of techniques result in detectable reporter gene activity, it remains to be proven if these techniques provide sufficient efficiency to produce biological effects from genes of interest. Challenges for the future include development of more efficient vectors and improved cell targeting. Despite these limitations, pulmonary vascular gene transfer holds promise as a useful investigative and therapeutic tool.
Approaching complexity: systems biology and ms-based techniques to address immune signaling
Published in Expert Review of Proteomics, 2020
Joseph Gillen, Caleb Bridgwater, Aleksandra Nita-Lazar
Reporter assays constitute the in vitro stimulation of a promoter and reporter gene pair to measure a response [2]. These assays can be extremely useful and are a robust method for high throughput screens of different drugs or compounds by the direct measurement of expressed gene products or the indirect changes in signal molecules regulated by genes. This is accomplished by transfecting a desired cell line to express a transcription factor activated promoter and a common reporter gene. The cells can then be stimulated to drive promoter activity. Ligand or agonist induction of the transcription factors leading to expression of the reporter gene. In the case of a luciferase reporter gene, luciferin, a luciferase substrate, can be added and the resulting luminescence measured as a corollary of stimulation. The unattractive variables of reporter assays tend to revolve around the limits in experimental design: reporter assays may take vast resources and long time periods to design and to target assays to transcription factors. Common reporter assays exploit the activity of reporter genes producing β-galactosidase, green fluorescent protein (GFP), and luciferase [2].
Promise of gene therapy to treat sickle cell disease
Published in Expert Opinion on Biological Therapy, 2018
Zulema Romero, Mark DeWitt, Mark C. Walters
In an alternative approach, Dever et al. [112] inserted a cDNA β-globin cassette at the start codon (flanked by 400 bp homology), so the expression of the cDNA is regulated by its endogenous promoter, delivered via AAV6. For this approach to be successful, the sgRNA editing site must be very near to the start codon to ensure that HDR is seamless and in frame after the start codon. In this construction, a reporter gene driven by its own promoter can be included to select for the modified cells. This strategy also has the potential to select for high-GFP expressing cells that will contain HBB-targeted HSPC. However, not all cellular markers are acceptable in a clinical setting, and there is a potential for enrichment of HSPC with proliferative characteristics introduced by off-target genomic modification. The truncated nerve growth factor receptor (tNGFR), (lacking its cytoplasmic intracellular signaling domain), has been be used to enrich for targeted HSPC by magnetic beads or FACS in clinical trials [117]. Dever et al. [112] used an anti-sickling cDNA expression cassette (AS3: Gly16Asp, Glu22Ala, Thr87Gln [58],) followed by the EF1α promoter driving tNGFR expression (HBB cDNA-EF1α-tNGFR). Using this AAV6 donor, AS3 mRNA expression was found in up to 56% of erythrocytes derived from SCD bone marrow cells, compared with 20% of the bulk population. However, optimization of a cDNA in lieu of the full genomic HBB donor template may be difficult. HBB mRNA stability and termination is highly dependent on its introns as was addressed previously in the section of LV design for SCD.
Synthetic biology-based portable in vitro diagnostic platforms
Published in Alexandria Journal of Medicine, 2018
Almando Geraldi, Ernawati Arifin Giri-Rachman
Here, we focus on two promising synthetic biology-inspired IVD platforms, Synthetic RNA- and CRISPR/Cas-based biosensors. Both methods detect and report the presence of pathogen via pathogen nucleic acid recognition. Synthetic RNA biosensor sensing module consists of RNA switch which contain sequences complementary to the target pathogen RNA. The binding of target RNA will activate the expression of reporter gene which product can be observed visually. While the CRISPR/Cas-based systems involve CRISPR RNA that bind to pathogen DNA or RNA and activate the non-specific activity of Cas nuclease to cleave quenched fluorescent reporter RNA. The cleaved RNA reporter will emit fluorescence signal which can be detected easily. Furthermore, the two systems can be applied to a paper-based platform which reduce operational costs, as well as simplify the storage and deployment in area with lack of advanced infrastructures and medical experts.