Mammalian Tissue Culture Research Articles

Identification of ERAD-dependent degrons for the endoplasmic reticulum lumen

Degrons are minimal protein features that are sufficient to target proteins for degradation. In most cases, degrons allow recognition by components of the cytosolic ubiquitin proteasome system. Currently, all of the identified degrons only function within the cytosol. Using Saccharomyces cerevisiae, we identified the first short linear sequences that function as degrons from the endoplasmic reticulum (ER) lumen. We show that when these degrons are transferred to proteins, they facilitate proteasomal degradation through the endoplasmic reticulum associated degradation (ERAD) system. These degrons enable degradation of both luminal and integral membrane ER proteins, expanding the types of proteins that can be targeted for degradation in budding yeast and mammalian tissue culture. This discovery provides a framework to target proteins for degradation from the previously unreachable ER lumen and builds toward therapeutic approaches that exploit the highly conserved ERAD system.

Corynebacterium bovis Growth in Tissue Culture Conditions and Media.

A common concern in preclinical cancer research is the introduction of Corynebacterium bovis into immunodeficient mouse colonies through cancer cell lines. C. bovis is a known contaminant of patient-derived xenograft tumors passaged horizontally between immunodeficient mice. However, it is unclear if C. bovis can grow in mammalian tissue culture conditions or tissue culture media. We hypothesized that C. bovis would not grow under tissue culture conditions or media, diminishing the risk of transmission from tumor cell lines cultured in vitro. Three C. bovis isolates, CUAMC1, HAC, and ATCC-7715, were used to test our hypothesis in 3 of the most common media used to grow human cancer cell lines including RPMI 1640 + 10% FBS (RPMI), DMEM/high glucose + 10% FBS (DMEM), and DMEM/F-12 + 10% FBS (DMEM/F12). Our results confirmed propagation of each C. bovis isolate in DMEM/F12 media under tissue culture conditions after 72 h. However, these results also demonstrate diminished viability of each C. bovis isolate in RPMI and DMEM after 72 h. To assess whether antibiotics could halt the growth of C. bovis under tissue culture conditions in DMEM/F12, penicillin-streptomycin (pen/strep) was added to the experimental media. This treatment was effective in eliminating all viable C. bovis in the culture system after 72 h. Our data suggest that C. bovis growth under tissue culture conditions is possible and growth in tissue culture media is nuanced. These results highlight the importance of pathogen surveillance for tumor cell lines propagated in vitro and demonstrate the need for further investigation into C. bovis growth requirements.

Identification of ERAD-dependent degrons for the endoplasmic reticulum lumen.

Degrons are minimal protein features that are sufficient to target proteins for degradation. In most cases, degrons allow recognition by components of the cytosolic ubiquitin proteasome system. Currently, all of the identified degrons only function within the cytosol. Using Saccharomyces cerevisiae, we identified the first short linear sequences that function as degrons from the endoplasmic reticulum (ER) lumen. We show that when these degrons are transferred to proteins, they facilitate proteasomal degradation through the ERAD system. These degrons enable degradation of both luminal and integral membrane ER proteins, expanding the types of proteins that can be targeted for degradation in budding yeast and mammalian tissue culture. This discovery provides a framework to target proteins for degradation from the previously unreachable ER lumen and builds toward therapeutic approaches that exploit the highly-conserved ERAD system.

Evidence for the Role of a Second Fc-Binding Receptor in Placental IgG Transfer in Nonhuman Primates.

Transplacental transfer of maternal antibodies provides the fetus and newborn with passive protection against infectious diseases. While the role of the highly conserved neonatal Fc receptor (FcRn) in transfer of IgG in mammals is undisputed, recent reports have suggested that a second receptor may contribute to transport in humans. We report poor transfer efficiency of plant-expressed recombinant HIV-specific antibodies, including engineered variants with high FcRn affinity, following subcutaneous infusion into rhesus macaques close to parturition. Unexpectedly, unlike those derived from mammalian tissue culture, plant-derived antibodies were essentially unable to cross macaque placentas. This defect was associated with poor Fcγ receptor binding and altered Fc glycans and was not recapitulated in mice. These results suggest that maternal-fetal transfer of IgG across the three-layer primate placenta may require a second receptor and suggest a means of providing maternal antibody treatments during pregnancy while avoiding fetal harm. IMPORTANCE This study compared the ability of several human HIV envelope-directed monoclonal antibodies produced in plants with the same antibodies produced in mammalian cells for their ability to cross monkey and mouse placentas. We found that the two types of antibodies have comparable transfer efficiencies in mice, but they are differentially transferred across macaque placentas, consistent with a two-receptor IgG transport model in primates. Importantly, plant-produced monoclonal antibodies have excellent binding characteristics for human FcRn receptors, permitting desirable pharmacokinetics in humans. The lack of efficient transfer across the primate placenta suggests that therapeutic plant-based antibody treatments against autoimmune diseases and cancer could be provided to the mother while avoiding transfer and preventing harm to the fetus.

A Suite of New Strain Construction Vectors for Gene Expression Knockdown in Budding Yeast.

Numerous tools for gene expression knockdown have been developed and characterized in the model organism Saccharomyces cerevisiae and extended to facilitate studies in multicellular models. To comparatively evaluate the efficacy of these approaches, we systematically applied seven such published constitutive and inducible knockdown strategies to a panel of essential genes encoding nuclear-localized proteins. In this effort, we created the CEAS (C-SWAT for Essential Allele Strains) collection, a suite of tagging vectors for improved utility and ease of strain construction. Of particular note, we adapted an improved auxin inducible degron (AID) protein degradation strategy previously available only in mammalian tissue culture for one-step strain construction in budding yeast by leveraging both the C-SWAT system and CRISPR/Cas9 editing. Taken together, this work presents a toolbox for endogenous gene expression knockdown and allows us to make recommendations on the efficacy and applicability of these tools for the perturbation of essential genes.

Polyethyleneimine/polyethylene glycol–conjugated gold nanoparticles as nanoscale positive/negative controls in nanotoxicology: testing in frog embryo teratogenesis assay–Xenopus and mammalian tissue culture system

Despite the great potential of using positively charged gold nanoparticles (AuNPs) in nanomedicine, no systematic studies have been reported on their synthesis optimization or colloidal stability under physiological conditions until a group at the National Institute of Standards and Technology recently succeeded in producing remarkably stable polyethyleneimine (PEI)-coated AuNPs (Au-PEI). This improved version of Au-PEI (Au-PEI25kB) has increased the demand for toxicity and teratogenicity information for applications in nanomedicine and nanotoxicology. In vitro assays for Au-PEI25kB in various cell lines showed substantial active cytotoxicity. For advanced toxicity research, the frog embryo teratogenesis assay–Xenopus (FETAX) method was employed in this study. We observed that positively-charged Au-PEI25kB exhibited significant toxicity and teratogenicity, whereas polyethylene glycol conjugated AuNPs (Au-PEG) used as comparable negative controls did not. There is a characteristic avidity of Au-PEI25kB for the jelly coat, the chorionic envelope (also known as vitelline membrane) and the cytoplasmic membrane, as well as a barrier effect of the chorionic envelope observed with Au-PEG. To circumvent these characteristics, an injection-mediated FETAX approach was utilized. Like treatment with the FETAX method, the injection of Au-PEI25kB severely impaired embryo development. Notably, the survival/concentration curve that was steep when the standard FETAX approach was employed became gradual in the injection-mediated FETAX. These results suggest that Au-PEI25kB may be a good candidate as a nanoscale positive control material for nanoparticle analysis in toxicology and teratology.

Anti‐Covid Gene Therapy: Integration of Authentic Research into a Continuously Evolving Undergraduate Laboratory Course

Undergraduate biology students often graduate without exposure to authentic research experiences. Laboratory courses follow a one or two week fail‐proof experiment resembling a cookbook recipe, lacking the uncertainty of genuine research. Techniques in molecular biology cover an array of skills essential to succeed in a biotechnological laboratory today. I have been developing an evolving molecular biology lab course based on the teaching of concepts while imparting the skills and applications of modern techniques, providing students with theoretical concepts and laboratory skills. We prepare students to carry‐out scientific protocols that can be applied to a future workforce setting. Students are immersed in a 10‐week series of labs with the objective to use molecular cloning to make a novel gene therapy vector; therapies are designed to inhibit the expression of genes of the virus that causes Covid‐19, severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2). Students are introduced to PubMed and GenBank to research the background of SARS‐CoV‐2 and its target genes; the Spike gene (S), the membrane gene (M) the nucleocapsid gene (N) and the envelope gene (E). Students use the DNA software, Serial Cloner, as a tool to evaluate DNA sequences and mFOLD to analyze RNA structure. Students generate and visualize the design of an antisense gene therapy directed against one of the target viral genes. Using our gene therapy platform, students generate a vector with a unique target. Subsequently student‐generated vectors were transfected into mammalian tissue culture cells that express one of the target genes (S, M, N or E) and RNA and protein was collected to measure the efficacy of the gene therapy vector to reduce the expression of the target viral gene. In our initial experiments, we observed greater than 2.5‐fold reduction in the Spike Covid mRNA. Currently, students are testing additional therapies to maximize efficacy.

Spatial and temporal control of mitochondrial H2O2 release in intact human cells

Hydrogen peroxide (H2O2) has key signaling roles at physiological levels, while causing molecular damage at elevated concentrations. H2O2 production by mitochondria is implicated in regulating processes inside and outside these organelles. However, it remains unclear whether and how mitochondria in intact cells release H2O2. Here, we employed a genetically encoded high‐affinity H2O2 sensor, HyPer7, in mammalian tissue culture cells to investigate different modes of mitochondrial H2O2 release. We found substantial heterogeneity of HyPer7 dynamics between individual cells. We further observed mitochondria‐released H2O2 directly at the surface of the organelle and in the bulk cytosol, but not in the nucleus or at the plasma membrane, pointing to steep gradients emanating from mitochondria. Gradient formation is controlled by cytosolic peroxiredoxins, which act redundantly and with a substantial reserve capacity. Dynamic adaptation of cytosolic thioredoxin reductase levels during metabolic changes results in improved H2O2 handling and explains previously observed differences between cell types. Our data suggest that H2O2‐mediated signaling is initiated only in close proximity to mitochondria and under specific metabolic conditions.

In vivo labeling of endogenous genomic loci in C. elegans using CRISPR/dCas9.

Visualization of genomic loci with open chromatin state has been reported in mammalian tissue culture cells using a CRISPR/Cas9-based system that utilizes an EGFP-tagged endonuclease-deficient Cas9 protein (dCas9::EGFP) (Chen et al. 2013). Here, we adapted this approach for use in Caenorhabditis elegans . We generated a C. elegans strain that expresses the dCas9 protein fused to two nuclear-localized EGFP molecules (dCas9::NLS::2xEGFP::NLS) in an inducible manner. Using this strain, we report the visualization in live C. elegans embryos of two endogenous repetitive loci, rrn-4 and rrn-1 , from which 5S and 18S ribosomal RNAs are constitutively generated.

Picroscope: low-cost system for simultaneous longitudinal biological imaging

Simultaneous longitudinal imaging across multiple conditions and replicates has been crucial for scientific studies aiming to understand biological processes and disease. Yet, imaging systems capable of accomplishing these tasks are economically unattainable for most academic and teaching laboratories around the world. Here, we propose the Picroscope, which is the first low-cost system for simultaneous longitudinal biological imaging made primarily using off-the-shelf and 3D-printed materials. The Picroscope is compatible with standard 24-well cell culture plates and captures 3D z-stack image data. The Picroscope can be controlled remotely, allowing for automatic imaging with minimal intervention from the investigator. Here, we use this system in a range of applications. We gathered longitudinal whole organism image data for frogs, zebrafish, and planaria worms. We also gathered image data inside an incubator to observe 2D monolayers and 3D mammalian tissue culture models. Using this tool, we can measure the behavior of entire organisms or individual cells over long-time periods.

Infection Kinetics and Transmissibility of a Reanimated Dengue Virus Serotype 4 Identified Originally in Wild Aedes aegypti From Florida.

Dengue virus is the most prevalent mosquito-borne virus, causing approximately 390 million infections and 25,000 deaths per year. Aedes aegypti, the primary mosquito vector of dengue virus, is well-established throughout the state of Florida, United States. Autochthonous transmission of dengue virus to humans in Florida has been increasing since 2009, alongside consistent importation of dengue cases. However, most cases of first infection with dengue are asymptomatic and the virus can be maintained in mosquito populations, complicating surveillance and leading to an underestimation of disease risk. Metagenomic sequencing of A. aegypti mosquitoes in Manatee County, Florida revealed the presence of dengue virus serotype 4 (DENV-4) genomes in mosquitoes from multiple trapping sites over 2years, in the absence of a human DENV-4 index case, and even though a locally acquired case of DENV-4 has never been reported in Florida. This finding suggested that: (i) DENV-4 may circulate among humans undetected; (ii) the virus was being maintained in the mosquito population, or (iii) the detected complete genome sequence may not represent a viable virus. This study demonstrates that an infectious clone generated from the Manatee County DENV-4 (DENV-4M) sequence is capable of infecting mammalian and insect tissue culture systems, as well as adult female A. aegypti mosquitoes when fed in a blood meal. However, the virus is subject to a dose dependent infection barrier in mosquitoes, and has a kinetic delay compared to a phylogenetically related wild-type (WT) control virus from a symptomatic child, DENV-4H (strain Homo sapiens/Haiti-0075/2015, GenBank accession MK514144.1). DENV-4M disseminates from the midgut to the ovary and saliva at 14days post-infection. Viral RNA was also detectable in the adult female offspring of DENV-4M infected mosquitoes. These results demonstrate that the virus is capable of infecting vector mosquitoes, is transmissible by bite, and is vertically transmitted, indicating a mechanism for maintenance in the environment without human-mosquito transmission. These findings suggest undetected human-mosquito transmission and/or long-term maintenance of the virus in the mosquito population is occurring in Florida, and underscore the importance of proactive surveillance for viruses in mosquitoes.GRAPHICAL ABSTRACTIn order to better assess the public health risk posed by a detection of DENV-4 RNA in Manatee County, FL Aedes aegypti, we produced an infectious clone using the sequence from the wild-caught mosquitoes and characterized it via laboratory infections of mosquitoes and mosquito tissues.

Intestinal organoid cocultures with microbes.

Adult-stem-cell-derived organoids model human epithelial tissues ex vivo, which enables the study of host-microbe interactions with great experimental control. This protocol comprises methods to coculture organoids with microbes, particularly focusing on human small intestinal and colon organoids exposed to individual bacterial species. Microinjection into the lumen and periphery of 3D organoids is discussed, as well as exposure of organoids to microbes in a 2D layer. We provide detailed protocols for characterizing the coculture with regard to bacterial and organoid cell viability and growth kinetics. Spatial relationships can be studied by fluorescence live microscopy, as well as scanning electron microscopy. Finally, we discuss considerations for assessing the impact of bacteria on gene expression and mutations through RNA and DNA sequencing. This protocol requires equipment for standard mammalian tissue culture, or bacterial or viral culture, as well as a microinjection device.

A genetically encoded tool for reconstituting synthetic modulatory neurotransmission and reconnect neural circuits in vivo

Chemogenetic and optogenetic tools have transformed the field of neuroscience by facilitating the examination and manipulation of existing circuits. Yet, the field lacks tools that enable rational rewiring of circuits via the creation or modification of synaptic relationships. Here we report the development of HySyn, a system designed to reconnect neural circuits in vivo by reconstituting synthetic modulatory neurotransmission. We demonstrate that genetically targeted expression of the two HySyn components, a Hydra-derived neuropeptide and its receptor, creates de novo neuromodulatory transmission in a mammalian neuronal tissue culture model and functionally rewires a behavioral circuit in vivo in the nematode Caenorhabditis elegans. HySyn can interface with existing optogenetic, chemogenetic and pharmacological approaches to functionally probe synaptic transmission, dissect neuropeptide signaling, or achieve targeted modulation of specific neural circuits and behaviors.

Practical Methods for Expression of Recombinant Protein in the Pichia pastoris System

One of the most critical challenges of genetic engineering is the expression of recombinant proteins using biological expression systems. Nowadays, different expression systems from bacteria to mammalian tissue culture cells are available for the production of recombinant proteins for medical and industrial purposes. Among various choices, yeast expression systems such as Pichia pastoris are promising candidates. The P. pastoris expression system is a standard tool for the production of biopharmaceuticals and industrial enzymes. It is also considered a unique host for the expression of subunit vaccines which could significantly affect the growing market of medical biotechnology. Using P. pastoris as an expression system for heterologous proteins, this article provides detailed basic protocols for cloning of a recombinant cassette into a suitable expression vector, the transformation of foreign vector DNAs into the yeast by electroporation, and expression and purification of desired recombinant protein. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Cloning of a recombinant cassette into a suitable expression vector Basic Protocol 2: Transformation of P. pastoris and selection of transformants Basic Protocol 3: Optimization and large-scale expression of recombinant proteins Basic Protocol 4: Purification of recombinant proteins.

Design, Synthesis and Testing of an Anti‐Covid Gene Therapy: Integration of Authentic Research into an Undergraduate Laboratory Course

Undergraduate biology students often graduate without exposure to authentic research experiences. Laboratory courses follow a one or two week fail-proof experiment resembling a cookbook recipe, lacking the uncertainty of genuine research. Techniques in molecular biology cover an array of skills essential to succeed in a biotechnological laboratory today. This lab course is based on the teaching of concepts while imparting the skills and applications of modern techniques, providing students with theoretical concepts and laboratory skills. We prepare students to carry-out scientific protocols that can be applied to a future workforce setting. Students are immersed in a 10-week series of labs with the objective to use molecular cloning to make a novel gene therapy vector; therapies are designed to inhibit the expression of genes of the virus that causes Covid-19, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Students are introduced to PubMed and Genbank to research the background of SARS-CoV-2 and its target genes; the Spike gene (S), the membrane gene (M) the nucleocapsid gene (N) and the envelope gene (E). Students use the DNA software, Serial Cloner, as a tool to evaluate DNA sequences and mFOLD to analyze RNA structure. Students generate and visualize the design of an antisense gene therapy directed against one of the target viral genes. Using our gene therapy platform, students generate a vector with a unique target. Subsequently student-generated vectors were transfected into mammalian tissue culture cells that express the target genes (S, M, N or E) and RNA and protein was collected to measure the efficacy of the gene therapy vector to reduce the expression of the target viral gene.

Stress Granule Protein Features Allow for Accurate Prediction of Biological Condensate Localization with GraPES

Biological condensates have been thrust to the forefront of molecular biology over the past decade for their implications in enzymatic activity, neurodegenerative diseases, and cellular organization. It is thought that many of these membraneless organelles are formed through a process known as liquid-liquid phase separation, in which key protein or nucleic acid drivers seed the formation of protein-rich foci within the cellular milieu. Stress granules are stress-induced assemblies that are belong to this group of biological condensates and are of keen interest to the biomolecular research community due to being linked to both long-term cell viability and a variety of protein aggregation-based diseases. Recently, a large amount of proteomic data has been generated that provides unprecedented insight into stress granule composition and stands as fruitful ground for further analysis. Interrogation of this data revealed that stress granule proteins are enriched in features that favor protein liquid-liquid phase separation. Proteins within stress granules were found to be more disordered, soluble, and abundant than their proteome and cytosolic controls while also having an increased potential for post-translational modifications. Furthermore, these “stress granuleomes” were found to be enriched for multivalent character by possessing multiple ordered domains and being likely to interact with RNA maintaining a high level of protein-protein interactions under basal conditions. Our findings are consistent with the notion that stress granule formation is driven by protein liquid-liquid phase separation. Furthermore, stress granule proteins appear poised near solubility limits while possessing the ability to dynamically alter their phase behavior in response to external threat. We culminate results from our analysis into novel predictors for granule incorporation in yeast and mammalian cells that out performs similar computational tools. Using this predictor, we were able to correctly identify new stress granule components, two of which we validated with colocalization microscopy in mammalian tissue culture cells [1]. We then developed a second series of sequence-base predictors and integrate all of these computational tools into a user-friendly web-based interface, called GraPES (Granule Protein Enrichment Server), where users can either look up a variety of pre-calculated likelihood z-scores for human and yeast proteins or obtain novel predictions from their own input FASTA formatted protein sequences.

In Vitro Cultivation of the Syphilis Spirochete Treponema pallidum.

For over a century, investigation of Treponema pallidum subsp. pallidum, the spiral‐shaped bacterium that causes syphilis, was hindered by an inability to culture the organism in vitro. A recent breakthrough has enabled continuous in vitro growth of this organism in co‐culture with mammalian tissue culture cells. This article contains the protocols needed to culture T. pallidum in the standard laboratory environment. In addition, protocols for growing and maintaining the required tissue culture cells, for generating isogenic strains by limiting dilution, and for quantitating T. pallidum by darkfield microscopy are included. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: In vitro cultivation of Treponema pallidum Basic Protocol 2: Generation of isogenic strains Support Protocol 1: Alternate harvest procedure Support Protocol 2: Culture of Sf1Ep cells Support Protocol 3: Assessment of T. pallidum number and viability

Methods for Characterization of Alternative RNA Splicing.

Quantification of alternative splicing to detect the abundance of differentially spliced isoforms of a gene in total RNA can be accomplished via RT-PCR using both quantitative real-time and semiquantitative PCR methods. These methods require careful PCR primer design to ensure specific detection of particular splice isoforms. We will also describe analysis of alternative splicing using a splicing "minigene" in mammalian cell tissue culture to facilitate investigation of the regulation of alternative splicing of a particular exon of interest.

Rapid Lentiviral Vector Producer Cell Line Generation Using a Single DNA Construct

Stable suspension producer cell lines for the production of vesicular stomatitis virus envelope glycoprotein (VSVg)-pseudotyped lentiviral vectors represent an attractive alternative to current widely used production methods based on transient transfection of adherent 293T cells with multiple plasmids. We report here a method to rapidly generate such producer cell lines from 293T cells by stable transfection of a single DNA construct encoding all lentiviral vector components. The resulting suspension cell lines yield titers as high as can be achieved with transient transfection, can be readily scaled up in single-use stirred-tank bioreactors, and are genetically and functionally stable in extended cell culture. By removing the requirement for efficient transient transfection during upstream processing of lentiviral vectors and switching to an inherently scalable suspension cell culture format, we believe that this approach will result in significantly higher batch yields than are possible with current manufacturing processes and enable better patient access to medicines based on lentiviral vectors.

Using unnatural amino acids to selectively label proteins for cellular imaging: a cell biologist viewpoint.

Twenty-five years ago, GFP revolutionized the field of cell biology by enabling scientists to visualize, for the first time, proteins in living cells. However, when it comes to current, state-of-the-art imaging technologies, fluorescent proteins (such as GFP) have several limitations that result from their size and photophysics. Over the past decade, an elegant, alternative approach, which is based on the direct labeling of proteins with fluorescent dyes and is compatible with live-cell and super-resolution imaging applications, has been introduced. In this approach, an unnatural amino acid that can covalently bind a fluorescent dye is incorporated into the coding sequence of a protein. The protein of interest is thereby site-specifically fluorescently labeled inside the cell, eliminating the need for protein- or peptide-labeling tags. Whether this labeling approach will change cell biology research is currently unclear, but it clearly has the potential to do so. In this short review, a general overview of this approach is provided, focusing on the imaging of site-specifically labeled proteins in mammalian tissue culture cells, and highlighting its advantages and limitations for cellular imaging.