Search for: All records

CRISPR/Cas9 gene editing is effective in manipulating genetic loci in mammalian cell cultures and whole fish but efficient platforms applicable to fish cell lines are currently limited. Our initial attempts to employ this technology in fish cell lines using heterologous promoters or a ribonucleoprotein approach failed to indicate genomic alteration at targeted sites in a tilapia brain cell line (OmB). For potential use in a DNA vector approach, endogenous tilapia beta Actin (OmBAct), EF1 alpha (OmEF1a), and U6 (TU6) promoters were isolated. The strongest candidate promoter determined by EGFP reporter assay, OmEF1a, was used to drive constitutive Cas9 expression in a modified OmB cell line (Cas9-OmB1). Cas9-OmB1 cell transfection with vectors expressing gRNAs driven by the TU6 promoter achieved mutational efficiencies as high as 81% following hygromycin selection. Mutations were not detected using human and zebrafish U6 promoters demonstrating the phylogenetic proximity of U6 promoters as critical when used for gRNA expression. Sequence alteration to TU6 improved mutation rate and cloning efficiency. In conclusion, we report new tools for ectopic expression and a highly efficient, economical system for manipulation of genomic loci and evaluation of their causal relationship with adaptive cellular phenotypes by CRISPR/Cas9 gene editing in fish cells.

Interactions of organisms with their environment are complex and regulation at different levels of biological organization from genotype to phenotype is often non-linear. While studies of transcriptome regulation are now common for many species, corresponding quantitative studies of environmental effects on proteomes are needed. Here we report the generation of a data-independent acquisition (DIA) assay library that enables simultaneous targeted proteomics of thousands of O. niloticus kidney proteins using a label- and gel-free workflow that is well suited for ecologically relevant field samples. Transcript and protein abundance differences in kidneys of tilapia acclimated to freshwater and brackish water (25 g/kg) were correlated for 2114 unique genes. A high degree of non-linearity in salinity-dependent regulation of transcriptomes and proteomes was revealed, demonstrating the complementary nature of the DIA assay library approach and suggesting that the regulation of O. niloticus renal function by environmental salinity relies heavily on post-transcriptional mechanisms. In addition to significance testing, the application of functional enrichment analyses using STRING and KEGG to DIA assay datasets identified myo-inositol metabolism, antioxidant and xenobiotic functions, and signaling mechanisms as key elements controlled by salinity in tilapia kidneys. In conclusion, this study presents an innovative approach for targeted quantitative proteomics used tomore »identify proteins and biological processes that are regulated non-linearly at mRNA and protein levels during a change of environmental salinity.« less

Interactions of organisms with their environment are complex and regulation at different levels of biological organization from genotype to phenotype is often non-linear. While studies of transcriptome regulation are now common for many species, corresponding quantitative studies of environmental effects on proteomes are needed. Here we report the generation of a data-independent acquisition (DIA) assay library that enables simultaneous targeted proteomics of thousands of O. niloticus kidney proteins using a label- and gel-free workflow that is well suited for ecologically relevant field samples. Transcript and protein abundance differences in kidneys of tilapia acclimated to freshwater and brackish water (25 g/kg) were correlated for 2114 unique genes. A high degree of non-linearity in salinity-dependent regulation of transcriptomes and proteomes was revealed, demonstrating the complementary nature of the DIA assay library approach and suggesting that the regulation of O. niloticus renal function by environmental salinity relies heavily on post-transcriptional mechanisms. In addition to significance testing, the application of functional enrichment analyses using STRING and KEGG to DIA assay datasets identified myo-inositol metabolism, antioxidant and xenobiotic functions, and signaling mechanisms as key elements controlled by salinity in tilapia kidneys. In conclusion, this study presents an innovative approach for targeted quantitative proteomics used tomore »identify proteins and biological processes that are regulated non-linearly at mRNA and protein levels during a change of environmental salinity.« less

Histone post-translational modifications (PTMs) are epigenetic marks that modify the state of chromatin and lead to alterations in gene expression. Advances in mass spectrometry have enabled the high-throughput analysis of histone PTMs without the need for prior knowledge of individual PTMs of interest. In this study, the global histone PTM landscape was analyzed in the gills, kidney, and testes of Mozambique tilapia (Oreochromis mossambicus) through tandem mass spectrometry using data dependent acquisition (DDA-LCMS2) and PTM mapping approaches. PTM assignment to a specific amino acid was validated using A-score and localization probability scores that are based on the detection of diagnostic MSMS ions. These values signify the robustness of PTM assignment to a specific residue within the protein sequence. For PTMs that were represented by both modified and unmodified versions of the corresponding peptide, the stoichiometry was calculated and compared between tissues. We have identified multiple types of histone PTMs and assigned them to specific residues in each tissue. These PTMs include acetylation, methylation, demethylation, trimethylation, phosphorylation/ dehydration, and ubiquitination. Our results indicate that the gills, kidney, and testes each display a unique profile of histone PTMs. These data provide a strong basis for the generation of spectral libraries that enablemore »high-throughput quantitative analyses of histone PTM stoichiometry on a global scale in tilapia exposed to diverse environmental and developmental contexts.« less

Cell cultures are effective supplemental models to study specific biochemical pathways used for environmental adaption in animals. They enable isolation from system influence and facilitate control the extracellular environment. For work focusing on fish species many representative cell lines now exist, including a tilapia brain cell line (OmB) developed in our lab. CRISPR/Cas9 gene editing is an additional tool aiding these studies by allowing manipulation of specific genetic loci and evaluating their causal relationship between phenotypes of interest. However, established CRISPR/Cas9 gene targeting tools and methods often have not functioned as efficiently in fish cells as seen in other animal cell models such as mammalian cell lines, consistent with our initial attempts to apply CRISPR/Cas9 in OmB cells that failed to indicate genomic alteration at the targeted sites. Poor expression of heterologous promoters in OmB cells was hypothesized to be a primary cause for this occurrence so we constructed a custom plasmid vector based system utilizing tilapia endogenous promoters (EF1 alpha to express Cas9 and a U6 to express gRNAs). This system demonstrated substantial editing of most target sites attempted with mutational efficiency as high 80%. This work specifically highlighted the importance of phylogenetic proximity in selection of a polymerasemore »III promoter for gRNA expression as commonly used interspecies U6 promoters (human and zebrafish) yielded no detectable gene editing when applied in this system with a common gRNA target sequence. These new tools will allow generation of knockout cell lines for gene targeting studies in tilapia and other phylogenetically close fish species.« less

Euryhaline fish tolerate a wide range of environmental salinity by employing molecular mechanisms for coping with the associated osmotic stress. We have previously shown that osmotic stress transcription factor 1 (OSTF1) is part of these mechanisms. OSTF1 is transiently and rapidly upregulated in gill epithelial cells of tilapia (Oreochromis mossambicus) exposed to hyperosmolality. Hyperosmotic induction of OSTF1 in tilapia gills was reproduced in the tilapia OmB cell neuroepithelial cell line. OSTF1 shares the signature sequence of the TSC-22 family suggesting that it is a transcriptional repressor. If, in fact, OSTF1 is a transcription factor, we hypothesize that it will localize to the nucleus during hyperosmotic stress. Using standard cloning procedures, OSTF1 was tagged with enhanced green fluorescent protein (EGFP) at either the C- or N-terminus. Using fluorescent microscopy we show that the fusion proteins are retained in the cytosol under iso-osmotic conditions. To evaluate potential nuclear translocation of OSTF1 during hyperosmotic stress, we subjected OmB cells expressing the OSTF1:EGFP fusion protein to hyperosmotic media and imaged at time intervals from 5 minutes to 4 hours using a Leica Dmi8 microscope with automated scanning stage. At four hours and 650 mOsmol/kg, subcellular localization quantified by LASX image analysis (Leica) demonstrated thatmore »OSTF1:EGFP was mostly localized to the nucleus. This result supports our hypothesis that OSTF1 is indeed an osmotically inducible transcription factor. Current work evaluates influence of specific OSTF1 domains on nuclear localization.« less

Using abundance measurements of 1,490 proteins from four separate populations of three-spined sticklebacks, we implemented a system-level approach to correlate proteome dynamics with environmental salinity and temperature and the fish's population and morphotype. We identified robust and accurate fingerprints that classify environmental salinity, temperature, morphotype, and the population sample origin, observing that proteins with specific functions are enriched in these fingerprints. Highly apparent functions represented in all fingerprints include ion transport, proteostasis, growth, and immunity, suggesting that these functions are most diversified in populations inhabiting different environments. Applying a differential network approach, we analyzed the network of protein interactions that differs between populations. Looking at specific population combinations of differential interaction, we identify sets of connected proteins. We find that these sets and their corresponding enriched functions reflect key processes that have diverged between the four populations. Moreover, the extent of divergence, i.e., the number of enriched functions that differ between populations, is highest when all three environmental parameters are different between two populations. Key nodes in the differential interaction network signify functions that are also inherent in the fingerprints, most prominently proteostasis-related functions. However, the differential interaction network also reveals additional functions that have diverged between populations, notably cytoskeletalmore »organization and morphogenesis. The strength of these analyses is that the results are purely data driven. With such an unbiased approach applied on a large proteomic data set, we find the strongest signals given by the data, making it possible to develop more discriminatory and complex biomarkers for specific contexts of interest.« less

Synopsis The gill proteome of threespine sticklebacks (Gasterosteus aculeatus) differs greatly in populations that inhabit diverse environments characterized by different temperature, salinity, food availability, parasites, and other parameters. To assess the contribution of a specific environmental parameter to such differences it is necessary to isolate its effects from those of other parameters. In this study the effect of environmental salinity on the gill proteome of G. aculeatus was isolated in controlled mesocosm experiments. Salinity-dependent changes in the gill proteome were analyzed by Liquid chromatography/Tandem mass spectrometry data-independent acquisition (DIA) and Skyline. Relative abundances of 1691 proteins representing the molecular phenotype of stickleback gills were quantified using previously developed MSMS spectral and assay libraries in combination with DIA quantitative proteomics. Non-directional stress responses were distinguished from osmoregulatory protein abundance changes by their consistent occurrence during both hypo- and hyper-osmotic salinity stress in six separate mesocosm experiments. If the abundance of a protein was consistently regulated in opposite directions by hyper- versus hypo-osmotic salinity stress, then it was considered an osmoregulatory protein. In contrast, if protein abundance was consistently increased irrespective of whether salinity was increased or decreased, then it was considered a non-directional response protein. KEGG pathway analysis revealed that themore »salivary secretion, inositol phosphate metabolism, valine, leucine, and isoleucine degradation, citrate cycle, oxidative phosphorylation, and corresponding endocrine and extracellular signaling pathways contain most of the osmoregulatory gill proteins whose abundance is directly proportional to environmental salinity. Most proteins that were inversely correlated with salinity map to KEGG pathways that represent proteostasis, immunity, and related intracellular signaling processes. Non-directional stress response proteins represent fatty and amino acid degradation, purine metabolism, focal adhesion, mRNA surveillance, phagosome, endocytosis, and associated intracellular signaling KEGG pathways. These results demonstrate that G. aculeatus responds to salinity changes by adjusting osmoregulatory mechanisms that are distinct from transient non-directional stress responses to control compatible osmolyte synthesis, transepithelial ion transport, and oxidative energy metabolism. Furthermore, this study establishes salinity as a key factor for causing the regulation of numerous proteins and KEGG pathways with established functions in proteostasis, immunity, and tissue remodeling. We conclude that the corresponding osmoregulatory gill proteins and KEGG pathways represent molecular phenotypes that promote transepithelial ion transport, cellular osmoregulation, and gill epithelial remodeling to adjust gill function to environmental salinity.« less