scholarly journals High-throughput subcellular protein localization using cell arrays

2005 ◽  
Vol 33 (6) ◽  
pp. 1407-1408 ◽  
Author(s):  
Y.-H. Hu ◽  
D. Vanhecke ◽  
H. Lehrach ◽  
M. Janitz
Keyword(s):  
High Throughput ◽  
Cellular Localization ◽  
Fluorescent Protein ◽  
Protein Localization ◽  
Open Reading Frames ◽  
Expression Vectors ◽  
Sequence Information ◽  
Cell Array ◽  
Single Experiment ◽  
Cell Arrays

Accomplishment of the human and mouse genome projects resulted in accumulation of extensive gene sequence information. However, the information about the biological functions of the identified genes remains a bottleneck of the post-genomic era. Hence, assays providing simple functional information, such as localization of the protein within the cell, can be very helpful in the elucidation of its function. Transfected cell arrays offer a robust platform for protein localization studies. Open reading frames of unknown genes can be linked to a His6-tag or GFP (green fluorescent protein) reporter in expression vectors and subsequently transfected using the cell array. Cellular localization of the transfected proteins is detected either by specific anti-His-tag antibodies or directly by fluorescence of the GFP fusion protein and by counterstaining with organelle-specific dyes. The high throughput of the method in terms of information provided for every single experiment makes this approach superior to classical immunohistological methods for protein localization.

scholarly journals High-Density Cell Arrays for Genome-Scale Phenotypic Screening

2019 ◽  
Vol 24 (3) ◽  
pp. 274-283 ◽  
Author(s):  
Vytaute Starkuviene ◽  
Stefan M. Kallenberger ◽  
Nina Beil ◽  
Tautvydas Lisauskas ◽  
Bastian So-Song Schumacher ◽  
...  
Keyword(s):  
New Technologies ◽  
High Density ◽  
Cell Array ◽  
Preclinical Development ◽  
Single Experiment ◽  
Efficient Manner ◽  
Cell Arrays ◽  
High Density Cell ◽  
High Degree ◽  
Genome Scale

Due to high associated costs and considerable time investments of cell-based screening, there is a strong demand for new technologies that enable preclinical development and tests of diverse biologicals in a cost-saving and time-efficient manner. For those reasons we developed the high-density cell array (HD-CA) platform, which miniaturizes cell-based screening in the form of preprinted and ready-to-run screening arrays. With the HD-CA technology, up to 24,576 samples can be tested in a single experiment, thereby saving costs and time for microscopy-based screening by 75%. Experiments on the scale of the entire human genome can be addressed in a real parallel manner, with screening campaigns becoming more comfortable and devoid of robotics infrastructure on the user side. The high degree of miniaturization enables working with expensive reagents and rare and difficult-to-obtain cell lines. We have also optimized an automated imaging procedure for HD-CA and demonstrate the applicability of HD-CA to CRISPR-Cas9- and RNAi-mediated phenotypic assessment of the gene function.

scholarly journals A Visual Screen of Protein Localization during Sporulation Identifies New Components of Prospore Membrane-Associated Complexes in Budding Yeast

2014 ◽  
Vol 13 (3) ◽  
pp. 383-391 ◽  
Author(s):  
Chien Lam ◽  
Ethan Santore ◽  
Elizabeth Lavoie ◽  
Leor Needleman ◽  
Nicholas Fiacco ◽  
...  
Keyword(s):  
Cellular Localization ◽  
Secretory Pathway ◽  
Fluorescent Protein ◽  
Protein Localization ◽  
Leading Edge ◽  
Ring Structure ◽  
Content Type ◽  
Prospore Membrane ◽  
Membrane Compartment ◽  
Green Fluorescent

ABSTRACT During ascospore formation in Saccharomyces cerevisiae , the secretory pathway is reorganized to create new intracellular compartments, termed prospore membranes. Prospore membranes engulf the nuclei produced by the meiotic divisions, giving rise to individual spores. The shape and growth of prospore membranes are constrained by cytoskeletal structures, such as septin proteins, that associate with the membranes. Green fluorescent protein (GFP) fusions to various proteins that associate with septins at the bud neck during vegetative growth as well as to proteins encoded by genes that are transcriptionally induced during sporulation were examined for their cellular localization during prospore membrane growth. We report localizations for over 100 different GFP fusions, including over 30 proteins localized to the prospore membrane compartment. In particular, the screen identified IRC10 as a new component of the leading-edge protein complex (LEP), a ring structure localized to the lip of the prospore membrane. Localization of Irc10 to the leading edge is dependent on SSP1 , but not ADY3 . Loss of IRC10 caused no obvious phenotype, but an ady3 irc10 mutant was completely defective in sporulation and displayed prospore membrane morphologies similar to those of an ssp1 strain. These results reveal the architecture of the LEP and provide insight into the evolution of this membrane-organizing complex.

scholarly journals High-throughput subcellular protein localization using cell arrays

2005 ◽  
Vol 33 (6) ◽  
pp. 1407 ◽  
Author(s):  
D. Vanhecke ◽  
Y.-H. Hu ◽  
H. Lehrach ◽  
M. Janitz
Keyword(s):  
High Throughput ◽  
Protein Localization ◽  
Subcellular Protein Localization ◽  
Cell Arrays

scholarly journals pFPL Vectors for High-Throughput Protein Localization in Fungi: Detecting Cytoplasmic Accumulation of Putative Effector Proteins

2015 ◽  
Vol 28 (2) ◽  
pp. 107-121 ◽  
Author(s):  
Xiaoyan Gong ◽  
Oscar Hurtado ◽  
Baohua Wang ◽  
Congqing Wu ◽  
Mihwa Yi ◽  
...  
Keyword(s):  
High Throughput ◽  
Large Scale ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Protein Localization ◽  
Yellow Fluorescent Protein ◽  
Effector Proteins ◽  
Fungal Transformation ◽  
In Planta ◽  
Green Fluorescent

As part of a large-scale project whose goal was to identify candidate effector proteins in Magnaporthe oryzae, we developed a suite of vectors that facilitate high-throughput protein localization experiments in fungi. These vectors utilize Gateway recombinational cloning to place a gene's promoter and coding sequences upstream and in frame with enhanced cyan fluorescent protein, green fluorescent protein (GFP), monomeric red fluorescence protein (mRFP), and yellow fluorescent protein or a nucleus-targeted mCHERRY variant. The respective Gateway cassettes were incorporated into Agrobacterium-based plasmids to allow efficient fungal transformation using hygromycin or geneticin resistance selection. mRFP proved to be more sensitive than the GFP spectral variants for monitoring proteins secreted in planta; and extensive testing showed that Gateway-derived fusion proteins produced localization patterns identical to their “directly fused” counterparts. Use of plasmid for fungal protein localization (pFPL) vectors with two different selectable markers provided a convenient way to label fungal cells with different fluorescent proteins. We demonstrate the utility of the pFPL vectors for identifying candidate effector proteins and we highlight a number of important factors that must be taken into consideration when screening for proteins that are translocated across the host plasma membrane.

scholarly journals Investigation Into the use of C- and N-terminal GFP Fusion Proteins for Subcellular Localization Studies Using Reverse Transfection Microarrays

2004 ◽  
Vol 5 (4) ◽  
pp. 342-353 ◽  
Author(s):  
Ella Palmer ◽  
Tom Freeman
Keyword(s):  
Subcellular Localization ◽  
High Throughput ◽  
Fusion Proteins ◽  
Fluorescent Protein ◽  
Protein Localization ◽  
Genes Encoding ◽  
Reverse Transfection ◽  
C And N ◽  
Green Fluorescent ◽  
Cellular Compartments

Reverse transfection microarrays were described recently as a high throughput method for studying gene function. We have investigated the use of this technology for determining the subcellular localization of proteins. Genes encoding 16 proteins with a variety of functions were placed in Gateway expression constructs with 3′ or 5′ green fluorescent protein (GFP) tags. These were then packaged in transfection reagent and spotted robotically onto a glass slide to form a reverse transfection array. HEK293T cells were grown over the surface of the array until confluent and GFP fluorescence visualized by confocal microscopy. All C-terminal fusion proteins localized to cellular compartments in accordance with previous studies and/or bioinformatic predictions. However, less than half of the N-terminal fusion proteins localized correctly. Of those that were not in concordance with the C-terminal tagged proteins, half did not exhibit expression and the remainder had differing subcellular localizations to the C-terminal fusion protein. This data indicates that N-terminal tagging with GFP adversely affects the protein localization in reverse transfection assays, whereas tagging with GFP at the C-terminal is generally better in preserving the localization of the native protein. We discuss these results in the context of developing high-throughput subcellular localization assays based on the reverse transfection array technology.

scholarly journals Expression of Foreign Proteins by Poliovirus Polyprotein Fusion: Analysis of Genetic Stability Reveals Rapid Deletions and Formation of Cardioviruslike Open Reading Frames

1998 ◽  
Vol 72 (1) ◽  
pp. 20-31 ◽  
Author(s):  
Steffen Mueller ◽  
Eckard Wimmer
Keyword(s):  
Fluorescent Protein ◽  
Growth Cycle ◽  
Open Reading Frames ◽  
Expression Vectors ◽  
Green Fluorescent Protein Gene ◽  
Published Data ◽  
Gene Encoding ◽  
Genomic Rnas ◽  
Reading Frame ◽  
Green Fluorescent

ABSTRACT Using a strategy developed by R. Andino, D. Silvera, S. D. Suggett, P. I. Achacoso, C. J. Miller, D. Baltimore, and M. B. Feinberg (Science 265:1448–1451, 1994), we constructed recombinant polioviruses by fusing the open reading frame (ORF) of the green fluorescent protein gene (gfp) of Aequorea victoria or the gag gene (encoding p17-p24) of human immunodeficiency virus type 1 (HIV-1) to the N terminus of the poliovirus polyprotein. All poliovirus expression vectors constructed by us and those obtained from Andino et al. were found to be severely impaired in viral replication and genetically unstable. Upon replication, inserted sequences were rapidly deleted as early as the first growth cycle in HeLa cells. However, the vector viruses did not readily revert to the wild-type sequence but rather retained some of the insert plus the artificial 3Cpro/3CDprocleavage site, engineered between the heterologous sequence and the poliovirus polyprotein, to give rise to genotypes reminiscent of cardioviruses. These virus variants that carry a small leader polypeptide were now relatively stable, and they grew better than their progenitor strains. Reverse transcription followed by PCR and sequence analysis of the genomic RNAs reproducibly revealed a few preferred genotypes among the isolated deletion variants. The remaining truncated inserts were retained through subsequent passages. In the immediate vicinity of the deletion borders, we observed short direct sequence repeats that we propose are involved in aligning RNA strands for illegitimate (nonhomologous) RNA recombination during minus-strand synthesis. On the basis of our results, which are at variance with published data, the utility of poliovirus vectors to express proteins >10 kDa in size through fusion with the polyprotein needs to be reevaluated.

scholarly journals Isolation of a Spodoptera exigua baculovirus recombinant with a 10·6 kbp genome deletion that retains biological activity

2000 ◽  
Vol 81 (10) ◽  
pp. 2545-2554 ◽  
Author(s):  
Xiaojiang Dai ◽  
József P. Hajós ◽  
Nina N. Joosten ◽  
Monique M. van Oers ◽  
Wilfred F. J. IJkel ◽  
...  
Keyword(s):  
Biological Activity ◽  
Spodoptera Exigua ◽  
Fluorescent Protein ◽  
Cultured Cells ◽  
Biological Activities ◽  
Biologically Active ◽  
Expression Vectors ◽  
Sequence Information ◽  
Protein Marker

When Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) is grown in insect cell culture, defective viruses are generated. These viruses lack about 25 kbp of sequence information and are no longer infectious for insects. This makes the engineering of SeMNPV for improved insecticidal activity or as expression vectors difficult to achieve. Recombinants of Autographa californica MNPV have been generated in insects after lipofection with viral DNA and a transfer vector into the haemocoel. In the present study a novel procedure to isolate SeMNPV recombinants was adopted by alternate cloning between insect larvae and cultured cells. The S. exigua cell line Se301 was used to select the putative recombinants by following a green fluorescent protein marker inserted in the p10 locus of SeMNPV. Polyhedra from individual plaques were fed to larvae to select for biological activity. In this way an SeMNPV recombinant (SeXD1) was obtained with the speed of kill improved by about 25%. This recombinant lacked 10593 bp of sequence information, located between 13·7 and 21·6 map units of SeMNPV and including ecdysteroid UDP glucosyl transferase, gp37, chitinase and cathepsin genes, as well as several genes unique to SeMNPV. The result indicated, however, that these genes are dispensable for virus replication both in vitro and in vivo. A mutant with a similar deletion was identified by PCR in the parental wild-type SeMNPV isolate, suggesting that genotypes with differential biological activities exist in field isolates of baculoviruses. The generation of recombinants in vivo, combined with the alternate cloning between insects and insect cells, is likely to be applicable to many baculovirus species in order to obtain biologically active recombinants.

scholarly journals Open Reading Frame E3-10.9K of Subspecies B1 Human Adenoviruses Encodes a Family of Late Orthologous Proteins That Vary in Their Predicted Structural Features and Subcellular Localization

2010 ◽  
Vol 84 (21) ◽  
pp. 11310-11322 ◽  
Author(s):  
Kathryn M. Frietze ◽  
Samuel K. Campos ◽  
Adriana E. Kajon
Keyword(s):  
Cellular Localization ◽  
Biochemical Characterization ◽  
Fluorescent Protein ◽  
Structural Features ◽  
Open Reading Frames ◽  
Late Time ◽  
Hydrophobic Domain ◽  
Reading Frame ◽  
Human Adenoviruses ◽  
Infected Cells

ABSTRACT Subspecies B1 human adenoviruses (HAdV-B1s) are important causative agents of acute respiratory disease, but the molecular bases of their distinct pathobiology are still poorly understood. Marked differences in genetic content between HAdV-B1s and the well-characterized HAdV-Cs that may contribute to distinct pathogenic properties map to the E3 region. Between the highly conserved E3-19K and E3-10.4K/RIDα open reading frames (ORFs), and in the same location as the HAdV-C ADP/E3-11.6K ORF, HAdV-B1s carry ORFs E3-20.1K and E3-20.5K and a polymorphic third ORF, designated E3-10.9K, that varies in the size of its predicted product among HAdV-B1 serotypes and genomic variants. As an initial effort to define the function of the E3-10.9K ORF, we carried out a biochemical characterization of E3-10.9K-encoded orthologous proteins and investigated their expression in infected cells. Sequence-based predictions suggested that E3-10.9K orthologs with a hydrophobic domain are integral membrane proteins. Ectopically expressed, C-terminally tagged (with enhanced green fluorescent protein [EGFP]) E3-10.9K and E3-9K localized primarily to the plasma membrane, while E3-7.7K localized primarily to a juxtanuclear compartment that could not be identified. EGFP fusion proteins with a hydrophobic domain were N and O glycosylated. EGFP-tagged E3-4.8K, which lacked the hydrophobic domain, displayed diffuse cellular localization similar to that of the EGFP control. E3-10.9K transcripts from the major late promoter were detected at late time points postinfection. A C-terminally hemagglutinin-tagged version of E3-9K was detected by immunoprecipitation at late times postinfection in the membrane fraction of mutant virus-infected cells. These data suggest a role for ORF E3-10.9K-encoded proteins at late stages of HAdV-B1 replication, with potentially important functional implications for the documented ORF polymorphism.

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