Harnessing Single Cell Sorting to Identify Cell Division Genes and Regulators in Bacteria

Cell division is an essential cellular process that requires an array of known and unknown proteins for its spatial and temporal regulation. Here we develop a novel, high-throughput screening method for the identification of bacterial cell division genes and regulators. The method combines the over-expression of a shotgun genomic expression library to perturb the cell division process with high-throughput flow cytometry sorting to screen many thousands of clones. Using this approach, we recovered clones with a filamentous morphology for the model bacterium, Escherichia coli. Genetic analysis revealed that our screen identified both known cell division genes, and genes that have not previously been identified to be involved in cell division. This novel screening strategy is applicable to a wide range of organisms, including pathogenic bacteria, where cell division genes and regulators are attractive drug targets for antibiotic development.     

A novel dual luciferase based high throughput assay to monitor autophagy in real time in yeast S. cerevisiae

BackgroundMacroautophagy is a cellular response to starvation wherein superfluous and damaged cytoplasmic constituents are degraded to provide energy for survival and to maintain cellular homeostasis. Dysfunctional autophagy is attributed to disease progression in several pathological conditions and therefore, autophagy has appeared as a potential pharmacological target for such conditions.ObjectiveIn search of potential drugs that modulate autophagy, identifying small molecule effectors of autophagy is the primary step. The conventional autophagy assays have a limitation that they cannot be scaled down to a high throughput format, therefore, novel sensitive assays are needed to discover new candidate molecules. Keeping this rationale in mind, a dual luciferase based assay was developed in the yeast S. cerevisiae that could measure both selective and general autophagy in real time.MethodsFirefly and Renilla luciferase reporter genes were cloned under POT-1 promoter. Using fatty acid medium the promoter was induced and the luciferase cargo was allowed to build up. The cells were then transferred to starvation conditions to stimulate autophagy and the degradation of luciferase markers was followed with time.Results and conclusionThe assay was more sensitive than conventional assays and could be scaled down to a 384 well format using an automated system. A good Z-factor score indicated that the assay is highly suitable for High Throughput Screening (HTS) of small molecule libraries. Screening of a small molecule library with our assay identified several known and novel modulators of autophagy.     

Optimized luciferase assay for cell-penetrating peptide-mediated delivery of short oligonucleotides

An improved assay for screening for the intracellular delivery efficacy of short oligonucleotides using cell-penetrating peptides is suggested. This assay is an improvement over previous assays that use luciferase reporters for cell-penetrating peptides because it has been scaled up from a 24-well format to a 96-well format and no longer relies on a luciferin reagent that has been commercially sourced. In addition, the homemade luciferin reagent is useful in multiple cell lines and in different assays that rely on altering the expression of luciferase. To establish a new protocol, the composition of the luciferin reagent was optimized for both signal strength and longevity by multiple two-factorial experiments varying the concentrations of adenosine triphosphate, luciferin, coenzyme A, and dithiothreitol. In addition, the optimal conditions with respect to cell number and time of transfection for both short interfering RNA (siRNA) and splice-correcting oligonucleotides (SCOs) are established. Optimal transfection of siRNA and SCOs was achieved using the reverse transfection method where the oligonucleotide complexes are already present in the wells before the cells are plated. Z′ scores were 0.73 for the siRNA assay and 0.71 for the SCO assay, indicating that both assays are suitable for high-throughput screening.     

Manipulation of the Porcine Epidemic Diarrhea Virus Genome Using Targeted RNA Recombination

Porcine epidemic diarrhea virus (PEDV) causes severe economic losses in the swine industry in China and other Asian countries. Infection usually leads to an acute, often lethal diarrhea in piglets. Despite the impact of the disease, no system is yet available to manipulate the viral genome which has severely hampered research on this virus until today. We have established a reverse genetics system for PEDV based on targeted RNA recombination that allows the modification of the 3′-end of the viral genome, which encodes the structural proteins and the ORF3 protein. Using this system, we deleted the ORF3 gene entirely from the viral genome and showed that the ORF3 protein is not essential for replication of the virus in vitro. In addition, we inserted heterologous genes (i.e. the GFP and Renilla luciferase genes) at two positions in the viral genome, either as an extra expression cassette or as a replacement for the ORF3 gene. We demonstrated the expression of both GFP and Renilla luciferase as well as the application of these viruses by establishing a convenient and rapid virus neutralization assay. The new PEDV reverse genetics system will enable functional studies of the structural proteins and the accessory ORF3 protein and will allow the rational design and development of next generation PEDV vaccines.     

High-Throughput Chemical Screening for Antivirulence Developmental Phenotypes in Trypanosoma brucei

In the bloodstream of mammalian hosts, the sleeping sickness parasite, Trypanosoma brucei, exists as a proliferative slender form or a nonproliferative, transmissible, stumpy form. The transition between these developmental forms is controlled by a density-dependent mechanism that is important for the parasite''s infection dynamics, immune evasion via ordered antigenic variation, and disease transmissibility. However, stumpy formation has been lost in most laboratory-adapted trypanosome lines, generating monomorphic parasites that proliferate uncontrolled as slender forms in vitro and in vivo. Nonetheless, these forms are readily amenable to cell culture and high-throughput screening for trypanocidal lead compounds. Here, we have developed and exploited a high-throughput screen for developmental phenotypes using a transgenic monomorphic cell line expressing a reporter under the regulation of gene control signals from the stumpy-specific molecule PAD1. Using a whole-cell fluorescence-based assay to screen over 6,000 small molecules from a kinase-focused compound library, small molecules able to activate stumpy-specific gene expression and proliferation arrest were assayed in a rapid assay format. Independent follow-up validation identified one hit able to induce modest, yet specific, changes in mRNA expression indicative of a partial differentiation to stumpy forms in monomorphs. Further, in pleomorphs this compound induced a stumpy-like phenotype, entailing growth arrest, morphological changes, PAD1 expression, and enhanced differentiation to procyclic forms. This not only provides a potential tool compound for the further understanding of stumpy formation but also demonstrates the use of high-throughput screening in the identification of compounds able to induce specific phenotypes, such as differentiation, in African trypanosomes.     

Dual Myxovirus Screen Identifies a Small-Molecule Agonist of the Host Antiviral Response

As we are confronted with an increasing number of emerging and reemerging viral pathogens, the identification of novel pathogen-specific and broad-spectrum antivirals has become a major developmental objective. Targeting of host factors required for virus replication presents a tangible approach toward obtaining novel hits with a broadened indication range. However, the identification of developable host-directed antiviral candidates remains challenging. We describe a novel screening protocol that interrogates the myxovirus host-pathogen interactome for broad-spectrum drug candidates and simultaneously probes for conventional, pathogen-directed hits. With resource efficiency and pan-myxovirus activity as the central developmental parameters, we explored coscreening against two distinct, independently traceable myxoviruses in a single-well setting. Having identified a pair of unrelated pathogenic myxoviruses (influenza A virus and measles virus) with comparable replication kinetics, we observed unimpaired coreplication of both viruses, generated suitable firefly and Renilla luciferase reporter constructs, respectively, and validated the protocol for up to a 384-well plate format. Combined with an independent counterscreen using a recombinant respiratory syncytial virus luciferase reporter, implementation of the protocol identified candidates with a broadened antimyxovirus profile, in addition to pathogen-specific hits. Mechanistic characterization revealed a newly discovered broad-spectrum lead that does not block viral entry but stimulates effector pathways of the innate cellular antiviral response. In summary, we provide proof of concept for the efficient discovery of broad-spectrum myxovirus inhibitors in parallel to para- and orthomyxovirus-specific hit candidates in a single screening campaign. The newly identified compound provides a basis for the development of a novel broad-spectrum small-molecule antiviral class.     

High-throughput proteomics: a flexible and efficient pipeline for protein production

Many studies that aim to characterize the proteome require the production of pure protein in a high-throughput format. We have developed a system for high-throughput subcloning, protein expression and purification that is simple, fast, and inexpensive. We utilized ligation-independent cloning with a custom-designed vector and developed an expression screen to test multiple parameters for optimal protein production in E. coli. A 96-well format purification protocol that produced microgram quantities of pure protein was also developed.     

A dual luciferase multiplexed high-throughput screening platform for protein-protein interactions

To study the biology of regulators of G-protein signaling (RGS) proteins and to facilitate the identification of small molecule modulators of RGS proteins, the authors recently developed an advanced yeast 2-hybrid (YTH) assay format for GalphaZ and RGS-Z1. Moreover, they describe the development of a multiplexed luciferase-based assay that has been successfully adapted to screen large numbers of small molecule modulators of protein-protein interactions. They generated and evaluated 2 different luciferase reporter gene systems for YTH interactions, a Gal4 responsive firefly luciferase reporter gene and a Gal4 responsive Renilla luciferase reporter gene. Both the firefly and Renilla luciferase reporter genes demonstrated a 40- to 50-fold increase in luminescence in strains expressing interacting YTH fusion proteins versus negative control strains. Because the firefly and Renilla luciferase proteins have different substrate specificity, the assays were multiplexed. The multiplexed luciferase-based YTH platform adds speed, sensitivity, simplicity, quantification, and efficiency to YTH high-throughput applications and therefore greatly facilitates the identification of small molecule modulators of protein-protein interactions as tools or potential leads for drug discovery efforts.     

An episomal expression vector for screening mutant gene libraries in Pichia pastoris

Screening mutant gene libraries for isolating improved enzyme variants is a powerful technique that benefits from effective and reliable biological expression systems. Pichia pastoris is a very useful organism to express proteins that are inactive in other hosts such as Escherichia coli and Saccharomyces cerevisiae. However, most P. pastoris expression plasmids are designed to integrate into the host chromosome and hence are not as amenable to high-throughput screening projects. We have designed a P. pastoris expression vector, pBGP1, incorporating an autonomous replication sequence that allows the plasmid to exist as an episomal element. This vector contains the alpha-factor signal sequence to direct secretion of the mutant enzymes. Expression of the genes is driven by the constitutive GAP promoter, thus eliminating the need for timed or cell density-specific inductions. The pBGP1 plasmid was used to screen a xylanase gene library to isolate higher activity mutants.     

Functional in vivo gene transfer into the myofibers of adult skeletal muscle

The postmitotic nature and longevity of skeletal muscle fibers permit stable expression of any transfected gene. Direct in vivo injection of plasmid DNA, in both adult and regenerating muscles, is a safe, inexpensive, and easy approach. Here we present an optimized electroporation protocol based on the use of spatula electrodes to transfer cDNA in vivo into the adult myofibers of an anatomically defined muscle, which could be functionally characterized. In our hands, about 80% of adult myofibers were transfected in vivo by different plasmids for GFP fusion proteins or for beta-galactosidase. The luciferase activity increased several orders of magnitude when compared to standard DNA delivery. In an anatomical defined muscle, the wide gene transfer was comparable to or better than that of retrovirus delivery, that recently has been shown to be prone to severe side-effects in human clinical studies. Furthermore, with our method the tissue damage was greatly decreased. Thus, the present work describes in vivo functional electrotransfer of genes in adult skeletal muscle fibers by a protocol that is of great potential for gene therapy, as well as for basic research.     

MISSION LentiPlex Pooled shRNA Library Screening in Mammalian Cells

RNA interference (RNAi) is an intrinsic cellular mechanism for the regulation of gene expression. Harnessing the innate power of this system enables us to knockdown gene expression levels in loss of gene function studies.There are two main methods for performing RNAi. The first is the use of small interfering RNAs (siRNAs) that are chemically synthesized, and the second utilizes short-hairpin RNAs (shRNAs) encoded within plasmids ¹. The latter can be transfected into cells directly or packaged into replication incompetent lentiviral particles. The main advantages of using lentiviral shRNAs is the ease of introduction into a wide variety of cell types, their ability to stably integrate into the genome for long term gene knockdown and selection, and their efficacy in conducting high-throughput loss of function screens. To facilitate this we have created the LentiPlex pooled shRNA library.The MISSION LentiPlex Human shRNA Pooled Library is a genome-wide lentiviral pool produced using a proprietary process. The library consists of over 75,000 shRNA constructs from the TRC collection targeting 15,000+ human genes ². Each library is tested for shRNA representation before product release to ensure robust library coverage. The library is provided in a ready-to-use lentiviral format at titers of at least 5 x 10⁸ TU/ml via p24 assay and is pre-divided into ten subpools of approximately 8,000 shRNA constructs each. Amplification and sequencing primers are also provided for downstream target identification.Previous studies established a synergistic antitumor activity of TRAIL when combined with Paclitaxel in A549 cells, a human lung carcinoma cell line ^{3, 4}. In this study we demonstrate the application of a pooled LentiPlex shRNA library to rapidly conduct a positive selection screen for genes involved in the cytotoxicity of A549 cells when exposed to TRAIL and Paclitaxel. One barrier often encountered with high-throughput screens is the cost and difficulty in deconvolution; we also detail a cost-effective polyclonal approach utilizing traditional sequencing.     

Up to 100-Fold Increase of Apparent Gene Expression in the Presence of Epstein-Barr Virus oriP Sequences and EBNA1: Implications of the Nuclear Import of Plasmids

A 100-fold increase in luciferase activity was observed in 293 cells, stably expressing Epstein-Barr nuclear antigen 1 (EBNA1; 293-EBNA1 cells), that had been transiently transfected with plasmids carrying Epstein-Barr virus (EBV) oriP sequences. This increase was observed in comparison to reporter gene activity obtained after transfection with a plasmid carrying no oriP sequences. The luciferase gene on these plasmids was under the control of either the cytomegalovirus immediate-early 1 gene enhancer-promoter (CMV IE1) or the Rous sarcoma virus promoter. The increase of reporter gene activity was not due to plasmid replication, since a similar enhancement was observed in the presence of aphidicolin, an inhibitor of replicative DNA synthesis, or after deletion of the dyad symmetry (DS) element within oriP. Luciferase production was not increased in the presence of only the DS element. Microinjection of plasmids carrying the CMV IE1 promoter-driven luciferase gene with or without oriP sequences into the nuclei of 293-EBNA1 cells resulted in a 17-fold increase in luciferase activity. Cytoplasmic injection of these plasmids led to an enhancement of luciferase activity of up to 100-fold. This difference in the factor of activation after nuclear or cytoplasmic injection could be ascribed to increased transport of plasmids carrying oriP from the cytoplasm to the nucleus in the presence of EBNA1. These data suggest the possibility of substantially increasing the apparent expression of a gene under the control of a strong constitutive promoter in the presence of oriP sequences and EBNA1. This improvement in expression is due to intranuclear enhancement of gene expression. oriP-specific transport of plasmid DNA from the cytoplasm of 293-EBNA1 cells to the nucleus seems to contribute to the observed effect.     

Rapid screening of a large insert BAC library for specific 16S rRNA genes using TRFLP

It is widely believed that the vast majority of microbes in the environment have-yet-to-be cultured using standard techniques. Bulk DNA from microbial communities is therefore often cloned into large insert vectors (e.g. bacterial artificial chromosomes [BAC] or cosmids) in order to study the genetic properties of these as yet (un)-cultured bacteria. In a typical BAC experiment, tens of thousands of clones are generated with only a small fraction of colonies containing the target(s) of interest. Efficient screening methodologies are therefore needed to allow targeted clone isolation. In this paper, we describe a rapid, inexpensive protocol that allows for the identification of specific 16S ribosomal RNA genes in a metagenomic library arrayed into 384-well microtiter plates. The rapid screening protocol employs Terminal Restriction Fragment Length Polymorphism (TRFLP) analysis to identify wells containing specific T-RF peaks. A nested approach using multiplexed samples of 384, 48, 8, and single colony analysis is described and applied in order to survey a BAC library generated from a marine microbial community off the coast of New Jersey. Screening revealed a total of 50 different 16 rRNA genes within the BAC library. Overall, the multiplexing format provided a simple, cost effective methodology for detecting clones bearing a target gene of interest in a large clone library. However, the limitations of screening BAC libraries using PCR methodologies and recommendations for improved screening efficiency using this approach are also discussed.     

Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes

We have developed a protocol to assemble in one step and one tube at least nine separate DNA fragments together into an acceptor vector, with 90% of recombinant clones obtained containing the desired construct. This protocol is based on the use of type IIs restriction enzymes and is performed by simply subjecting a mix of 10 undigested input plasmids (nine insert plasmids and the acceptor vector) to a restriction-ligation and transforming the resulting mix in competent cells. The efficiency of this protocol allows generating libraries of recombinant genes by combining in one reaction several fragment sets prepared from different parental templates. As an example, we have applied this strategy for shuffling of trypsinogen from three parental templates (bovine cationic trypsinogen, bovine anionic trypsinogen and human cationic trypsinogen) each divided in 9 separate modules. We show that one round of shuffling using the 27 trypsinogen entry plasmids can easily produce the 19,683 different possible combinations in one single restriction-ligation and that expression screening of a subset of the library allows identification of variants that can lead to higher expression levels of trypsin activity. This protocol, that we call ‘Golden Gate shuffling’, is robust, simple and efficient, can be performed with templates that have no homology, and can be combined with other shuffling protocols in order to introduce any variation in any part of a given gene.     

Cell-based screening and validation of human novel genes associated with cell viability

In the present study, a cell-based high-throughput assay is established to identify novel human genes associated with cell viability. The assay relies on the down-regulation of Renilla luciferase (pRL) activity in a 96-well format. In addition, 2-color fluorescence probes were used to distinguish living and dead cells. As the positive control, the authors used the expression vectors encoding Bax, TNFRSF1A, and TAJ, which were widely known to effectively induce programmed cell death. They screened 409 novel genes (including alternative mRNA splicing forms) cloned in their laboratory and found that 39 genes could significantly down-regulate pRL activity. A subsequent fluorescence-based assay revealed that 4 of the 39 genes (PIP5KL1, OLFM1, RNF122, FAM26B) were associated with cell viability. Further function assays validated that the 4 genes were able to induce both necrosis and apoptosis. These results therefore indicate that a rapid and effective screening system has been developed, which should shed light on some functions of novel genes.