Mammalian Cell Systems Research Articles

Using Synthetically Engineered Guide RNAs to Enhance CRISPR Genome Editing Systems in Mammalian Cells.

CRISPR-Cas9 is quickly revolutionizing the way we approach gene therapy. CRISPR-Cas9 is a complexed, two-component system using a short guide RNA (gRNA) sequence to direct the Cas9 endonuclease to the target site. Modifying the gRNA independent of the Cas9 protein confers ease and flexibility to improve the CRISPR-Cas9 system as a genome-editing tool. gRNAs have been engineered to improve the CRISPR system's overall stability, specificity, safety, and versatility. gRNAs have been modified to increase their stability to guard against nuclease degradation, thereby enhancing their efficiency. Additionally, guide specificity has been improved by limiting off-target editing. Synthetic gRNA has been shown to ameliorate inflammatory signaling caused by the CRISPR system, thereby limiting immunogenicity and toxicity in edited mammalian cells. Furthermore, through conjugation with exogenous donor DNA, engineered gRNAs have been shown to improve homology-directed repair (HDR) efficiency by ensuring donor proximity to the edited site. Lastly, synthetic gRNAs attached to fluorescent labels have been developed to enable highly specific nuclear staining and imaging, enabling mechanistic studies of chromosomal dynamics and genomic mapping. Continued work on chemical modification and optimization of synthetic gRNAs will undoubtedly lead to clinical and therapeutic benefits and, ultimately, routinely performed CRISPR-based therapies.

Process economics evaluation of cell-free synthesis for the commercial manufacture of antibody drug conjugates.

Continuous improvements of cell-free synthesis (CFS) systems have generated interest in adopting the technology for the manufacture of biologics. This paper provides an evaluation of the manufacturing cost-effectiveness of CFS for the commercial production of antibody-drug conjugates (ADCs). The evaluation was performed using an advanced techno-economic engine (TEE) built in Python. The TEE is programmed in an object-oriented environment capable of simulating a plethora of process flowsheets and predicting size and cost metrics for the process and the facility. A case study was formulated to compare the economics of whole bioprocesses based on either a CFS system or a mammalian cell system (CHO) for the manufacture of an ADC at a range of product demands. The analysis demonstrated the potential of CFS for the commercial manufacture of biologics and identified key cost drivers related to the system. The CFS system showed an approximately 80% increase in the cost of goods compared to CHO with a significant cost attributed to the in-house manufacture of the bacterial cell extract, necessary for the CFS reaction step in the process. A sensitivity and target analysis highlighted the need for further process improvements especially in the titer for the CFS process to become more competitive against well-established systems.

Expression and Purification of Yeast-derived GPCR, Gα and Gβγ Subunits for Structural and Dynamic Studies.

In the last several years, as evidence of a surged number of GPCR-G complex structures, the expressions of GPCRs and G proteins for structural biology have achieved tremendous successes, mostly in insect and mammalian cell systems, resulting in more than 370 structures of over 70 GPCRs have been resolved. However, the challenge remains, particularly in the conformational transition and dynamics study area where a much higher quantity of the receptors and G proteins is required even in comparison to X-ray and cryo-EM (5 mg/ml, 3 μl/sample) when NMR spectroscopy (5 mg/ml, 250 μl /sample) is applied. As a result, the expression levels of the insect and mammalian systems are also difficult to meet this demand, not to mention the prohibitive cost of producing GPCRs and G proteins using these systems for a vast majority of laboratories. Therefore, exploration of an effective, affordable, and practical approach with broad applicability is demanded. Pichia pastoris expression system has shown its promise in the GPCR preparation with many merits that other eukaryotic expression systems can't compete with. GPCRs expressed in this system are inexpensive, easy-to-manipulate, and capable of isotopically labeling. Herein, we present related protocols recently developed and upgraded in our lab, including expressions and purifications of P. pastoris derived GPCR along with Gα and Gβγ proteins. We anticipate that these protocols will advance the conformational transition and dynamics studies of the GPCR and its complexes.

Effect of Vedic music on steroidogenic gene expression in 3D-cultured buffalo granulosa cell spheroids model system, a pilot study.

Music is known for reducing stress, anxiety and depression, improving cognitive performance, and enhancing oestrogen levels. However, its effect on non-auditory mammalian cell system and the molecular events leading to higher oestrogen levels is less explored. Therefore, the present study targeted to know the direct effects of a peaceful Vedic music on 3D cultured buffalo granulosa cell spheroids. The spheroids were daily exposed to the Mahamrityunjaya mantra, a kind of Vedic chants, for 1.5hr for 6days. After 6days, the music effect was analysed by the expression analysis of steroidogenic (CYP19A1, STAR and HSD17β1) and proliferative marker (PCNA) genes. Interestingly, the CYP19A1 gene expression was significantly upregulated by 3.464±0.15 folds in the music exposed spheroids than the non-exposed spheroids. However, the expression of other steroidogenic and proliferative genes was unaltered. These observations provided a transcriptional clue for higher estradiol levels by the music and a scope to use Vedic chants for increasing the CYP19A1 expression to help tackle some pathophysiological conditions.

Modeling human brain tumors in flies, worms, and zebrafish: From proof of principle to novel therapeutic targets

For decades, cell biologists and cancer researchers have taken advantage of non-murine species to increase our understanding of the molecular processes that drive normal cell and tissue development, and when perturbed, cause cancer. The advent of whole-genome sequencing has revealed the high genetic homology of these organisms to humans. Seminal studies in non-murine organisms such as Drosophila melanogaster, Caenorhabditis elegans, and Danio rerio identified many of the signaling pathways involved in cancer. Studies in these organisms offer distinct advantages over mammalian cell or murine systems. Compared to murine models, these three species have shorter lifespans, are less resource intense, and are amenable to high-throughput drug and RNA interference screening to test a myriad of promising drugs against novel targets. In this review, we introduce species-specific breeding strategies, highlight the advantages of modeling brain tumors in each non-mammalian species, and underscore the successes attributed to scientific investigation using these models. We conclude with an optimistic proposal that discoveries in the fields of cancer research, and in particular neuro-oncology, may be expedited using these powerful screening tools and strategies.

Ubiquitin conjugating enzymes in the regulation of the autophagy-dependent degradation pathway

The ubiquitin-proteasomal system and the autophagy-lysosome system are two major degradation systems in mammalian cells. Ubiquitin not only regulates proteasomal degradation of substrates but also regulates the autophagy pathway. In one type of macroautophagy, called selective autophagy, cargos are recruited to phagophore in a ubiquitin-dependent manner. Ubiquitin can target autophagy regulators for proteasomal degradation, control protein conformation or change interacting partners of these regulators. To understand the regulatory mechanisms of these degradation pathways, it is critical to dissect how the ubiquitin system contributes to them. Since enzymes are key regulators of ubiquitination, in this review, such enzymes in autophagy regulation are discussed, with specific focus on ubiquitin conjugating enzyme E2s, of which roles in autophagy are emerging.

Structural and functional comparison of SARS-CoV-2-spike receptor binding domain produced in Pichia pastoris and mammalian cells

The yeast Pichia pastoris is a cost-effective and easily scalable system for recombinant protein production. In this work we compared the conformation of the receptor binding domain (RBD) from severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) Spike protein expressed in P. pastoris and in the well established HEK-293T mammalian cell system. RBD obtained from both yeast and mammalian cells was properly folded, as indicated by UV-absorption, circular dichroism and tryptophan fluorescence. They also had similar stability, as indicated by temperature-induced unfolding (observed Tm were 50 °C and 52 °C for RBD produced in P. pastoris and HEK-293T cells, respectively). Moreover, the stability of both variants was similarly reduced when the ionic strength was increased, in agreement with a computational analysis predicting that a set of ionic interactions may stabilize RBD structure. Further characterization by high-performance liquid chromatography, size-exclusion chromatography and mass spectrometry revealed a higher heterogeneity of RBD expressed in P. pastoris relative to that produced in HEK-293T cells, which disappeared after enzymatic removal of glycans. The production of RBD in P. pastoris was scaled-up in a bioreactor, with yields above 45 mg/L of 90% pure protein, thus potentially allowing large scale immunizations to produce neutralizing antibodies, as well as the large scale production of serological tests for SARS-CoV-2.

Optimization of Light-Inducible Gal4/UAS Gene Expression System in Mammalian Cells.

SummaryLight-inducible gene expression systems represent powerful methods for studying the functional roles of dynamic gene expression. Here, we developed an optimized light-inducible Gal4/UAS gene expression system for mammalian cells. We designed photoactivatable (PA)-Gal4 transcriptional activators based on the concept of split transcription factors, in which light-dependent interactions between Cry2-CIB1 PA-protein interaction modules can reconstitute a split Gal4 DNA-binding domain and p65 transcription activation domain. We developed a set of PA-Gal4 transcriptional activators (PA-Gal4cc), which differ in terms of induced gene expression levels following pulsed or prolonged light exposure, and which have different activation/deactivation kinetics. These systems offer optogenetic tools for the precise manipulation of gene expression at fine spatiotemporal resolution in mammalian cells.

N-Terminal-Dependent Protein Degradation and Targeting Cancer Cells.

Intracellular protein degradation is mediated selectively by the Ubiquitin-Proteasome System (UPS) and autophagic-lysosomal system in mammalian cells. Many cellular and physiological processes, such as cell division, cell differentiation, and cellular demise, are fine-tuned via the UPS-mediated protein degradation. Notably, impairment of UPS contributes to human disorders, including cancer and neurodegeneration. The proteasome- dependent N-degron pathways mediate the degradation of proteins through their destabilizing aminoterminal residues. Recent advances unveiled that targeting N-degron proteolytic pathways can aid in sensitizing some cancer cells to chemotherapeutic agents. Furthermore, interestingly, exploiting the N-degron feature, the simplest degradation signal in mammals, and fusing it to a ligand specific for Estrogen-Related Receptor alpha (ERRa) has demonstrated its utility in ERRa knockdown, via N-terminal dependent degradation, and also its efficiency in the inhibition of growth of breast cancer cells. These recent advances uncover the therapeutic implications of targeting and exploiting N-degron proteolytic pathways to curb growth and migration of cancer cells.

A versatile genetic control system in mammalian cells and mice responsive to clinically licensed sodium ferulate.

Dynamically adjustable gene- and cell-based therapies are recognized as next-generation medicine. However, the translation of precision therapies into clinics is limited by lack of specific switches controlled by inducers that are safe and ready for clinical use. Ferulic acid (FA) is a phytochemical with a wide range of therapeutic effects, and its salt sodium ferulate (SF) is used as an antithrombotic drug in clinics. Here, we describe an FA/SF-adjustable transcriptional switch controlled by the clinically licensed drug SF. We demonstrated that SF-responsive switches can be engineered to control CRISPR-Cas9 systems for on-command genome/epigenome engineering. In addition, we integrated FA-controlled switches into programmable biocomputers to process logic operations. We further demonstrated the dose-dependent SF-inducible transgene expression in mice by oral administration of SF tablets. Engineered switches responsive to small-molecule clinically licensed drugs to achieve adjustable transgene expression profiles provide new opportunities for dynamic interventions in gene- and cell-based precision medicine.

Dynamic regulation of NADPH oxidase 5 by intracellular heme levels and cellular chaperones

NADPH oxidase 5 (NOX5) is a transmembrane signaling enzyme that produces superoxide in response to elevated cytosolic calcium. In addition to its association with numerous human diseases, NOX5 has recently been discovered to play crucial roles in the immune response and cardiovascular system. Details of NOX5 maturation, and specifically its response to changes in intracellular heme levels have remained unclear. Here we establish an experimental system in mammalian cells that allows us to probe the influence of heme availability on ROS production by NOX5. We identified a mode of dynamic regulatory control over NOX5 activity through modulation of its heme saturation and oligomeric state by intracellular heme levels and Hsp90 binding. This regulatory mechanism allows for fine-tuning and reversible modulation of NOX5 activity in response to stimuli.

Identification of novel activators of the metal responsive transcription factor (MTF-1) using a gene expression biomarker in a microarray compendium.

Environmental exposure to metals is known to cause a number of human toxicities including cancer. Metal-responsive transcription factor 1 (MTF-1) is an important component of metal regulation systems in mammalian cells. Here, we describe a novel method to identify chemicals that activate MTF-1 based on microarray profiling data. MTF-1 biomarker genes were identified that exhibited consistent, robust expression across 10 microarray comparisons examining the effects of metals (zinc, nickel, lead, arsenic, mercury, and silver) on gene expression in human cells. A subset of the resulting 81 biomarker genes was shown to be altered by knockdown of the MTF1 gene including metallothionein family members and a zinc transporter. The ability to correctly identify treatment conditions that activate MTF-1 was determined by comparing the biomarker to microarray comparisons from cells exposed to reference metal activators of MTF-1 using the rank-based Running Fisher algorithm. The balanced accuracy for prediction was 93%. The biomarker was then used to identify organic chemicals that activate MTF-1 from a compendium of 11 725 human gene expression comparisons representing 2582 chemicals. There were 700 chemicals identified that included those known to interact with cellular metals, such as clioquinol and disulfiram, as well as a set of novel chemicals. All nine of the novel chemicals selected for validation were confirmed to activate MTF-1 biomarker genes in MCF-7 cells and to lesser extents in MTF1-null cells by qPCR and targeted RNA-Seq. Overall, our work demonstrates that the biomarker for MTF-1 coupled with the Running Fisher test is a reliable strategy to identify novel chemical modulators of metal homeostasis using gene expression profiling.

Involvement of a Na+-coupled Oligopeptide Transport System for β-amyloid Peptide (Aβ1–42) in Brain Cells

A Na+-coupled transport system in mammalian cells is responsible for the uptake of oligopeptides consisting of 5 or more amino acids. Here we investigated if this transport system is expressed in brain cells and transports the 42-amino-acid β-amyloid peptide (Aβ1-42). The human and mouse neuronal cell lines SK-N-SH and HT22, human microglial cell line HMC-3, and human blood-brain barrier endothelial cell line hCMEC/D3 were used to monitor the uptake of [3H]-deltorphin II (a heptapeptide) and fluorescence-labeled Aβ1-42. All four cell lines exhibited Na+-coupled uptake of deltorphin II. Aβ1-42 competed with deltorphin II for the uptake. Uptake of fluorescence-labeled Aβ1-42 was detectable in these cell lines, and the uptake was Na+-dependent and inhibitable by deltorphin II. The Na+-coupled uptake disappeared at high concentrations of Aβ1-42 due to oligomerization of the peptide. Exposure of the cells to excess iron abolished the uptake. In hCMEC/D3 cells cultured on Transwell filters, the uptake was localized preferentially to the abluminal membrane. A Na+-coupled transport system mediates the uptake of Aβ1-42 monomers in neuronal and microglial cells. The same system is also responsible for the uptake of Aβ1-42 from brain into blood-brain barrier endothelial cells. These findings have relevance to Alzheimer's disease.

Host-pathogen transcriptomics: Trypanosoma cruzi as a model for studying RNA contamination

Cellular infection assays constitute essential tools to understand host-pathogen interactions, particularly for intracellular microorganisms that are produced in cell lines are needed to propagate the microorganism. In this work, we demonstrate that RNA derived from Vero cells is an important contaminant to consider in order to avoid false positive results in transcriptomic experiments. We study the cross-contamination on a Trypanosoma cruzi cell infection model, the etiological agent of Chagas disease. We implemented the most frequently used trypanosome-purification protocols and, for all of them, we detected RNAs derived from Vero cells in trypomastigote extracts. For some of the protocols we also detected Vero RNAs in infected human cells. We also found this type of contamination in microarray experiments of human samples infected with T. cruzi. Concerning Illumina RNA-Seq data, we found that the contamination with Vero cells is probably introducing spurious results. Finally, we recommend a protocol to purify trypomastigotes, which showed a high percentage of trypomastigote recovery and the absence of Vero contamination in infected human samples. Avoiding this type of contamination should be an important factor to consider during experimental design, in order to minimize false positive results in transcriptomic studies as well as RNA contamination in vaccine production. SignificanceTranscriptomic studies are widely used to understand host-pathogen interactions. When the pathogen is an intracellular microorganism, an additional mammalian cell system can be needed to propagate it. In this work we demonstrate that pathogens purified from infected monolayers can carry RNAs from these mammalian cells, and that this ambient RNA contamination is probably producing false positive results in subsequent transcriptomic studies performed with qRT-PCR, microarrays or Next Generation Sequencing.

Expression vector cassette engineering for recombinant therapeutic production in mammalian cell systems.

Human tissue plasminogen activator was the first recombinant therapy protein that successfully produced in Chinese hamster ovary cells in 1986 and approved for clinical use. Since then, more and more therapeutic proteins are being manufactured in mammalian cells, and the technologies for recombinant protein production in this expression system have developed rapidly, with the optimization of both upstream and downstream processes. One of the most promising strategies is expression vector cassette optimization based on the expression vector cassette. In this review paper, these approaches and developments are summarized, and the future strategy on the utilizing of expression cassettes for the production of recombinant therapeutic proteins in mammalian cells is discussed.

RNA Interference Screening Identifies Novel Roles for RhoBTB1 and RhoBTB3 in Membrane Trafficking Events in Mammalian Cells.

In the endomembrane system of mammalian cells, membrane traffic processes require a high degree of regulation in order to ensure their specificity. The range of molecules that participate in trafficking events is truly vast, and much attention to date has been given to the Rab family of small GTPases. However, in recent years, a role in membrane traffic for members of the Rho GTPase family, in particular Cdc42, has emerged. This prompted us to develop and apply an image-based high-content screen, initially focussing on the Golgi complex, using RNA interference to systematically perturb each of the 21 Rho family members and assess their importance to the overall organisation of this organelle. Analysis of our data revealed previously unreported roles for two atypical Rho family members, RhoBTB1 and RhoBTB3, in membrane traffic events. We find that depletion of RhoBTB3 affects the morphology of the Golgi complex and causes changes in the trafficking speeds of carriers operating at the interface of the Golgi and endoplasmic reticulum. In addition, RhoBTB3 was found to be present on these carriers. Depletion of RhoBTB1 was also found to cause a disturbance to the Golgi architecture, however, this phenotype seems to be linked to endocytosis and retrograde traffic pathways. RhoBTB1 was found to be associated with early endosomal intermediates, and changes in the levels of RhoBTB1 not only caused profound changes to the organisation and distribution of endosomes and lysosomes, but also resulted in defects in the delivery of two different classes of cargo molecules to downstream compartments. Together, our data reveal new roles for these atypical Rho family members in the endomembrane system.

Anti-CRISPR protein applications: natural brakes for CRISPR-Cas technologies.

Clustered, regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) genes, a diverse family of prokaryotic adaptive immune systems, have emerged as a biotechnological tool and therapeutic. The discovery of protein inhibitors of CRISPR-Cas systems, called anti-CRISPR (Acr) proteins, enables the development of more controllable and precise CRISPR-Cas tools. Here we discuss applications of Acr proteins for post-translational control of CRISPR-Cas systems in prokaryotic and mammalian cells, organisms and ecosystems.

Dynamic Environmental Control in Microfluidic Single-Cell Cultivations: From Concepts to Applications.

Microfluidic single-cell cultivation (MSCC) is an emerging field within fundamental as well as applied biology. During the last years, most MSCCs were performed at constant environmental conditions. Recently, MSCC at oscillating and dynamic environmental conditions has started to gain significant interest in the research community for the investigation of cellular behavior. Herein, an overview of this topic is given and microfluidic concepts that enable oscillating and dynamic control of environmental conditions with a focus on medium conditions are discussed, and their application in single-cell research for the cultivation of both mammalian and microbial cell systems is demonstrated. Furthermore, perspectives for performing MSCC at complex dynamic environmental profiles of single parameters and multiparameters (e.g., pH and O2 ) in amplitude and time are discussed. The technical progress in this field provides completely new experimental approaches and lays the foundation for systematic analysis of cellular metabolism at fluctuating environments.

Synthetic mRNA-Based Systems in Mammalian Cells.

Living organisms are programmed to perform multiple functions by sensing intra- and extra-cellular environments and by controlling gene expressions. Synthetic biologists aim to program cells by mimicking, designing, and constructing genetic circuits. Synthetic mRNA-based genetic switches and circuits have attracted attention for future therapeutic applications because of their safety and functional diversity. Here, the mRNA-based switches and circuits that detect specific microRNAs or proteins expressed in a target cell to control transgene expression and cell fate are reviewed. Future perspectives of artificial RNA systems for cell engineering will also be addressed.

Mutual Regulation of RNA Silencing and the IFN Response as an Antiviral Defense System in Mammalian Cells.

RNA silencing is a posttranscriptional gene silencing mechanism directed by endogenous small non-coding RNAs called microRNAs (miRNAs). By contrast, the type-I interferon (IFN) response is an innate immune response induced by exogenous RNAs, such as viral RNAs. Endogenous and exogenous RNAs have typical structural features and are recognized accurately by specific RNA-binding proteins in each pathway. In mammalian cells, both RNA silencing and the IFN response are induced by double-stranded RNAs (dsRNAs) in the cytoplasm, but have long been considered two independent pathways. However, recent reports have shed light on crosstalk between the two pathways, which are mutually regulated by protein–protein interactions triggered by viral infection. This review provides brief overviews of RNA silencing and the IFN response and an outline of the molecular mechanism of their crosstalk and its biological implications. Crosstalk between RNA silencing and the IFN response may reveal a novel antiviral defense system that is regulated by miRNAs in mammalian cells.