Fluorescence-based phenotypic selection allows forward genetic screens in haploid human cells

The isolation of haploid cell lines has recently allowed the power of forward genetic screens to be applied to mammalian cells. The interest in applying this powerful genetic approach to a mammalian system is only tempered by the limited utility of these screens, if confined to lethal phenotypes. Here we expand the scope of these approaches beyond live/dead screens and show that selection for a cell surface phenotype via fluorescence-activated cell sorting can identify the key molecules in an intracellular pathway, in this case MHC class I antigen presentation. Non-lethal haploid genetic screens are widely applicable to identify genes involved in essentially any cellular pathway.     

High-content imaging-based pooled CRISPR screens in mammalian cells

CRISPR (clustered regularly interspaced short palindromic repeats)-based gene inactivation provides a powerful means for linking genes to particular cellular phenotypes. CRISPR-based screening typically uses large genomic pools of single guide RNAs (sgRNAs). However, this approach is limited to phenotypes that can be enriched by chemical selection or FACS sorting. Here, we developed a microscopy-based approach, which we name optical enrichment, to select cells displaying a particular CRISPR-induced phenotype by automated imaging-based computation, mark them by photoactivation of an expressed photoactivatable fluorescent protein, and then isolate the fluorescent cells using fluorescence-activated cell sorting (FACS). A plugin was developed for the open source software μManager to automate the phenotypic identification and photoactivation of cells, allowing ∼1.5 million individual cells to be screened in 8 h. We used this approach to screen 6,092 sgRNAs targeting 544 genes for their effects on nuclear size regulation and identified 14 bona fide hits. These results present a scalable approach to facilitate imaging-based pooled CRISPR screens.     

Bacterial surface display library screening by target enzyme labeling: Identification of new human cathepsin G inhibitors

The aim of this study was to establish a new tool for screening surface displayed peptide libraries based on the idea that cells expressing an enzyme inhibitor at the surface can be specifically labeled by the target enzyme. For this purpose peptide P15, exhibiting a K(i) value of 0.25 microM toward human cathepsin G, was expressed on the Escherichia coli cell surface by the use of Autodisplay. Purified cathepsin G was coupled to biotin and incubated with cells expressing the inhibitor. After addition of streptavidin-fluorescein isothiocyanate, these cells could be clearly differentiated from control cells by whole-cell fluorescence using flow cytometer analysis. To determine whether this protocol can be used for the sorting of single cells, a mixed population of cells with and without inhibitor was treated accordingly. Single cells were selected by increased fluorescence and sorted using fluorescence-activated cell sorting (FACS). Single cell clones were obtained and subjected to DNA sequence analysis. It turned out that the bacteria selected by this protocol displayed the correct peptide inhibitor at the cell surface. The protocol was then used to screen random peptide libraries, expressed at the cell surface, and a new lead structure for human cathepsin G (IC50 = 11.7 microM) was identified. The new drug discovery tool presented here consists of three steps: (a) surface display of peptide libraries, (b) selection of single cells with inhibiting structures by using the inherent affinity of the target enzyme, and (c) sorting of single cells, which were labeled by FACS.     

Embryonic and induced pluripotent stem cell staining and sorting with the live-cell fluorescence imaging probe CDy1

Detecting and isolating specific types of cells is crucial to understanding a variety of biological processes, including development, aging, regeneration and pathogenesis; this understanding, in turn, allows the use of cells for therapeutic purposes, for which stem cells have emerged recently as invaluable materials. The current methods of isolation and characterization of stem cells depend on cell morphology in culture or on immunostaining of specific markers. These methods are, however, time consuming and involve the use of antibodies that may often make the cells unsuitable for further study. We recently developed a fluorescent small molecule named CDy1 (compound of designation yellow 1) that selectively stains live embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). This protocol describes detailed procedures for staining ESC and iPSC in live conditions and for fluorescence-activated cell sorting (FACS) of ESC using CDy1. Cell staining, image acquisition and FACS can be done within 6 h.     

Messenger RNA quantification after fluorescence activated cell sorting using intracellular antigens

Recent studies using stem cells or cancer stem cells have revealed the importance of detecting minor populations of cells in blood or tissue and analyzing their biological characteristics. The only possible method for carrying out such procedures is fluorescence activated cell sorting (FACS). However, FACS has the following limitations. First, cells without an appropriate cell surface marker cannot be sorted. Second, the cells have to be kept alive during the sorting process in order to analyze their biological characteristics. If an intracellular antigen that was specific to a particular cell type could be stained with a florescent dye and then the cells can be sorted without causing RNA degradation, a more simple and universal method for sorting and analyzing cells with a specific gene expression pattern could be established since the biological characteristics of the sorted cells could then be determined by analyzing their gene expression profile. In this study, we established a basic protocol for messenger RNA quantification after FACS (FACS-mQ) targeting intracellular antigens. This method can be used for the detection and analysis of stem cells or cancer stem cells in various tissues.     

Flow sorting from organ material by intracellular markers.

BACKGROUND: Fluorescence-activated cell sorting (FACS) is an attractive technique for gene or protein expression studies in rare cell populations. For cell types where specific surface markers are not known, intracellular markers can be used. However, this approach is currently held to be difficult, as the required fixation and permeabilization may cause protein modification and RNA degradation. METHODS AND RESULTS: Using the rat thyroid gland as model, rare (parafollicular) and frequent (follicular) endocrine cell types were sorted based on immunostaining for intracellular calcitonin peptide and thyroglobulin protein expression. The sorted cells were compatible with Western blot analysis of proteins, immunoassay detection of calcitonin peptide hormone and RT-PCR. CONCLUSION: We developed a robust FACS protocol that allows flow sorting of rare cells from dissociated organ material, based on intracellular markers. Our FACS protocol is compatible with downstream analysis of proteins, peptides, and mRNA in the sorted cells.     

Green fluorescent protein as a second selectable marker for selection of high producing clones from transfected CHO cells

Mammalian cells are often used for the expression of recombinant proteins. The process of screening transfected cells randomly for high producing clones is tedious and time consuming. We evaluated using green fluorescent protein (GFP) for selection of high producing clones by fluorescence-activated cell sorter (FACS) to reduce screening effort. We expressed neurotrophin-3 (NT3), deoxyribonuclease (DNase), or vascular endothelial growth factor (VEGF) with GFP in Chinese hamster ovary cells. The vector expressed the desired secreted protein and the selectable marker, dihydrofolate reductase, in one expression unit and the intracellular GFP in a second expression unit. Transfected cells were grown in selection medium and sorted by FACS. High fluorescence clones were obtained and found to produce high amounts of the desired protein; VEGF productivity correlated well with GFP fluorescence in 48 clones. Further studies demonstrated that productivity correlated very well with RNA of the desired protein. For comparison, we randomly picked and screened 144 VEGF clones, and the highest producing VEGF clone obtained produced 0.7 pg/cell/day. In contrast, the highest producing VEGF clone obtained by FACS sorting produced 4.4 pg/cell/day. FACS sorting therefore selected high producing clones efficiently. Since an assay for the desired protein is not required, high producing clones for a protein of unknown function can be obtained by FACS sorting followed by measuring the RNA level of the desired protein in the highly fluorescent clones.     

Accelerated cell line development using two-color fluorescence activated cell sorting to select highly expressing antibody-producing clones

The success of engineered monoclonal antibodies as biopharmaceuticals has generated considerable interest in strategies designed to accelerate development of antibody expressing cell lines. Stable mammalian cell lines that express therapeutic antibodies at high levels typically take 6-12 months to develop. Here we describe a novel method to accelerate selection of cells expressing recombinant proteins (e.g., antibodies) using multiparameter fluorescence activated cell sorting (FACS) in association with dual intracellular autofluorescent reporter proteins. The method is co-factor-independent and does not require complex sample preparation. Chinese hamster ovary (CHO) clones expressing high levels of recombinant antibody were selected on the basis of a two-color FACS sorting strategy using heavy and light chain-specific fluorescent reporter proteins. We were able to establish within 12 weeks of transfection cell lines with greater than a 38-fold increase in antibody production when compared to the pool from which they were isolated, following a single round of FACS. The method provides a robust strategy to accelerate selection and characterization of clones and builds a foundation for a predictive model of specific productivity based upon on two-color fluorescence.     

A cell sorting protocol for selecting high-producing sub-populations of Sf9 and High Five™ cells

Insect cell lines such as Sf9 and High Five™ have been widely used to produce recombinant proteins mostly by the lytic baculovirus vector system. We have recently established an expression platform in Sf9 cells using a fluorescence-based recombinase mediated cassette exchange (RMCE) strategy which has similar development timelines but avoids baculovirus infection. To expedite cell engineering efforts, a robust fluorescence-activated cell sorting (FACS) protocol optimized for insect cells was developed here. The standard sorting conditions used for mammalian cells proved to be unsuitable, resulting in post-sorting viabilities below 10% for both cell lines. We found that the extreme sensitivity to the shear stress displayed by Sf9 and High Five™ cells was the limiting factor, and using Pluronic F-68 in the cell suspension could increase post-sorting viabilities in a dose dependent manner. The newly developed protocol was then used to sort stable populations of both cell lines tagged with a DsRed-expressing cassette. Before sorting, the average fluorescence intensity of the Sf9 cell population was 3-fold higher than that of the High Five™ cell population. By enriching with the 10% strongest DsRed-fluorescent cells, the productivity of both cell populations could be successfully improved. The established sorting protocol potentiates the use of RMCE technology for recombinant protein production in insect cells.     

Enrichment of slow-growing marine microorganisms from mixed cultures using gel microdrop (GMD) growth assay and fluorescence-activated cell sorting

Encapsulation of cells in agarose gel microdrops (GMDs) combined with fluorescence-activated cell sorting (FACS) has been used previously to analyze and recover specific mammalian, bacterial, and yeast cell populations. Recently, we have developed a method to enrich mixed bacterial populations for slow-growing microorganisms using the GMD Growth Assay combined with fluorochrome staining and flow cytometry. Here, we demonstrate the feasibility of using this experimental approach to detect clonogenic growth of individual bacteria within GMDs in less than 3 h and to separate subpopulations based on differential growth rates. We show that after sorting, organisms remain viable and can be propagated in culture for further analysis.     

Counting mutagenized genomes and optimizing genetic screens in Caenorhabditis elegans

In genetic screens, the number of mutagenized gametes examined is an important parameter for evaluating screen progress, the number of genes of a given mutable phenotype, gene size, cost, and labor. Since genetic screens often entail examination of thousands or tens of thousands of animals, strategies for optimizing genetics screens are important for minimizing effort while maximizing the number of mutagenized gametes examined. To date, such strategies have not been described for genetic screens in the nematode Caenorhabditis elegans. Here we review general principles of genetic screens in C. elegans, and use a modified binomial strategy to obtain a general expression for the number of mutagenized gametes examined in a genetic screen. We use this expression to calculate optimal screening parameters for a large range of genetic screen types. In addition, we developed a simple online genetic-screen-optimization tool that can be used independently of this paper. Our results demonstrate that choosing the optimal F2-to-F1 screening ratio can significantly improve screen efficiency.     

Axon regeneration mechanisms: insights from C. elegans

Understanding the mechanisms of axon regeneration is of great importance to the development of therapeutic treatments for spinal cord injury or stroke. Axon regeneration has long been studied in diverse vertebrate and invertebrate models, but until recently had not been analyzed in the genetically tractable model organism Caenorhabditis elegans. The small size, simple neuroanatomy, and transparency of C. elegans allows single fluorescently labeled axons to be severed in live animals using laser microsurgery. Many neurons in C. elegans are capable of regenerative regrowth, and can in some cases re-establish functional connections. Large-scale genetic screens have begun to elucidate the genetic basis of axon regrowth.     

Microplate-based high throughput screening procedure for the isolation of lipid-rich marine microalgae

ABSTRACT: We describe a new selection method based on BODIPY (4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene) staining, fluorescence activated cell sorting (FACS) and microplate-based isolation of lipid-rich microalgae from an environmental sample. Our results show that direct sorting onto solid medium upon FACS can save about 3 weeks during the scale-up process as compared with the growth of the same cultures in liquid medium. This approach enabled us to isolate a biodiverse collection of several axenic and unialgal cultures of different phyla.     

Combined Flow Cytometric Analysis of Surface and Intracellular Antigens Reveals Surface Molecule Markers of Human Neuropoiesis

Surface molecule profiles undergo dynamic changes in physiology and pathology, serve as markers of cellular state and phenotype and can be exploited for cell selection strategies and diagnostics. The isolation of well-defined cell subsets is needed for in vivo and in vitro applications in stem cell biology. In this technical report, we present an approach for defining a subset of interest in a mixed cell population by flow cytometric detection of intracellular antigens. We have developed a fully validated protocol that enables the co-detection of cluster of differentiation (CD) surface antigens on fixed, permeabilized neural cell populations defined by intracellular staining. Determining the degree of co-expression of surface marker candidates with intracellular target population markers (nestin, MAP2, doublecortin, TUJ1) on neuroblastoma cell lines (SH-SY5Y, BE(2)-M17) yielded a combinatorial CD49f^-/CD200^high surface marker panel. Its application in fluorescence-activated cell sorting (FACS) generated enriched neuronal cultures from differentiated cell suspensions derived from human induced pluripotent stem cells. Our data underlines the feasibility of using the described co-labeling protocol and co-expression analysis for quantitative assays in mammalian neurobiology and for screening approaches to identify much needed surface markers in stem cell biology.     

Engineering of staphylococcal surfaces for biotechnological applications

Novel surface proteins can be introduced onto bacterial cell surfaces by recombinant means. Here, we describe various applications of two such display systems for the food-grade bacteria Staphylococcus carnosus and Staphylococcus xylosus, respectively. The achievements in the use of such staphylococci as live bacterial vaccine delivery vehicles will be described. Co-display of proteins and peptides with adhesive properties to enable targeting of the bacteria, have significantly improved the vaccine delivery potential. Recently, protective immunity to respiratory syncytial virus (RSV) could be evoked in mice by intranasal immunization using such 'second generation' vaccine delivery systems. Furthermore, antibody fragments and other 'affinity proteins' with capacity to specifically bind a certain protein, e.g. Staphylococcus aureus protein A-based affibodies, have been surface-displayed on staphylococci as initial efforts to create whole-cell diagnostic devices. Surface display of metal-binding peptides, or protein domains into which metal binding properties has been engineered by combinatorial protein engineering, have been exploited to create staphylococcal bioadsorbents for potential environmental or biosensor applications. The use of these staphylococcal surface display systems as alternatives for display of large protein libraries and subsequent affinity selection of relevant binding proteins by fluorescence-activated cell sorting (FACS) will be discussed.     

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS)

Experimental and clinical studies often require highly purified cell populations. FACS is a technique of choice to purify cell populations of known phenotype. Other bulk methods of purification include panning, complement depletion and magnetic bead separation. However, FACS has several advantages over other available methods. FACS is the preferred method when very high purity of the desired population is required, when the target cell population expresses a very low level of the identifying marker or when cell populations require separation based on differential marker density. In addition, FACS is the only available purification technique to isolate cells based on internal staining or intracellular protein expression, such as a genetically modified fluorescent protein marker. FACS allows the purification of individual cells based on size, granularity and fluorescence. In order to purify cells of interest, they are first stained with fluorescently-tagged monoclonal antibodies (mAb), which recognize specific surface markers on the desired cell population (1). Negative selection of unstained cells is also possible. FACS purification requires a flow cytometer with sorting capacity and the appropriate software. For FACS, cells in suspension are passed as a stream in droplets with each containing a single cell in front of a laser. The fluorescence detection system detects cells of interest based on predetermined fluorescent parameters of the cells. The instrument applies a charge to the droplet containing a cell of interest and an electrostatic deflection system facilitates collection of the charged droplets into appropriate collection tubes (2). The success of staining and thereby sorting depends largely on the selection of the identifying markers and the choice of mAb. Sorting parameters can be adjusted depending on the requirement of purity and yield. Although FACS requires specialized equipment and personnel training, it is the method of choice for isolation of highly purified cell populations.     

Efficient generation of stably electrotransfected human hematopoietic cell lines without drug selection by consecutive FACsorting

BACKGROUND: Current methods to establish stably transfected cell lines by nonviral techniques involve coselection for a drug selection marker. However, this approach suffers from several drawbacks. We developed a fluorescence-activated cell sorting (FACS)-based protocol for the selection and isolation of stable hematopoietic electrotransfectants without the need for selective growth conditions. METHODS: Leukemic K562 cells were electroporated with the enhanced green fluorescent protein (EGFP) reporter gene and FACsorted to obtain stably EGFP-expressing cells. Stable EGFP(+) clones were established by single-cell sorting. RESULTS: Efficiency of stable EGFP gene expression increased steadily in function of number of consecutive FACsorts. Stable transfectants (>99% EGFP(+)) were obtained after four FACsorts. Furthermore, several single-cell derived clones with variable levels of stable EGFP expression were isolated and cultured without the use of selective growth media. CONCLUSIONS: EGFP is an effective selection marker for the generation and isolation of stably transfected hematopoietic cell clones without the need for selection in toxic media that could create a potentially undesirable stress environment for stably transfected cells.     

Rapid phenotypic analysis of uncoated Drosophila samples with low-vacuum scanning electron microscopy

Research projects featuring repetitive phenotypic analysis of insects, such as taxonomic studies, quantitative genetics, and mutant screens, could be greatly facilitated by a simpler approach to scanning electron microscopy (SEM). Here, we have applied low-vacuum SEM to wild type and mutant Drosophila and demonstrate that high quality ultrastructure data can be obtained quickly using minimal preparation. Adult flies, frozen live for storage, were mounted on aluminum stubs with carbon cement and directly imaged, with no chemical treatment or sputter coating. The key imaging parameters were identified and optimized, including chamber pressure, beam size, accelerating voltage, working distance and beam exposure. Different optimal conditions were found for eyes, wings, and bristles; in particular, surface features of bristles were obscured at higher accelerating voltages. The chief difficulties were charging, beam damage, and sample movement. We conclude that our optimized protocol is well suited to large-scale ultrastructural phenotypic analysis in insects.