Alternative splicing plays a critical role in the epithelial-mesenchymal transition (EMT), an essential cellular program that occurs in various physiological and pathological processes. Here we describe a strategy to detect alternative splicing during EMT using an inducible EMT model by expressing the transcription repressor Twist. EMT is monitored by changes in cell morphology, loss of E-cadherin localization at cell-cell junctions, and the switched expression of EMT markers, such as loss of epithelial markers E-cadherin and γ-catenin and gain of mesenchymal markers N-cadherin and vimentin. Using isoform-specific primer sets, the alternative splicing of interested mRNAs are analyzed by quantitative RT-PCR. The production of corresponding protein isoforms is validated by immunoblotting assays. The method of detecting splice isoforms described here is also suitable for the study of alternative splicing in other biological processes.
20 Related JoVE Articles!
Small-scale Nuclear Extracts for Functional Assays of Gene-expression Machineries
Institutions: Harvard Medical School.
A great deal of progress in understanding gene expression has been made using in vitro
systems. For most studies, functional assays are carried out using extracts that are prepared in bulk from 10-50 or more liters of cells grown in suspension. However, these large-scale preparations are not amenable to rapidly testing in vitro
effects that result from a variety of in vivo
cellular treatments or conditions. This journal video article shows a method for preparing functional small-scale nuclear extracts, using HeLa cells as an example. This method is carried out using as few as three 150 mm plates of cells grown as adherent monolayers. To illustrate the efficiency of the small-scale extracts, we show that they are as active as bulk nuclear extracts for coupled RNA Polymerase II transcription/splicing reactions. To demonstrate the utility of the extract protocol, we show that splicing is abolished in extracts prepared from HeLa cells treated with the splicing inhibitor drug E7107. The small-scale protocol should be generally applicable to any process or cell type that can be investigated in vitro
using cellular extracts. These include patient cells that are only available in limited quantities or cells exposed to numerous agents such as drugs, DNA damaging agents, RNAi, or transfection, which require the use of small cell populations. In addition, small amounts of freshly grown cells are convenient and/or required for some applications.
Cellular Biology, Issue 64, Genetics, HeLa nuclear extract, small-scale extract, pre-mRNA splicing, RNA polymerase II transcription, RNAi, coupled transcription/splicing, in vitro gene expression assays
Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection
Institutions: University of Toronto.
In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although the nuclear export of mRNA has been studied extensively in Xenopus oocytes1
and genetically tractable organisms such as yeast2
and the Drosophila
derived S2 cell line3
, few studies had been conducted in mammalian cells. Furthermore the kinetics of mRNA export in mammalian somatic cells could only be inferred indirectly4,5
. In order to measure the nuclear export kinetics of mRNA in mammalian tissue culture cells, we have developed an assay that employs the power of microinjection coupled with fluorescent in situ
hybridization (FISH). These assays have been used to demonstrate that in mammalian cells, the majority of mRNAs are exported in a splicing dependent manner6,7
, or in manner that requires specific RNA sequences such as the signal sequence coding region (SSCR) 6
. In this assay, cells are microinjected with either in vitro
synthesized mRNA or plasmid DNA containing the gene of interest. The microinjected cells are incubated for various time points then fixed and the sub-cellular localization of RNA is assessed using FISH. In contrast to transfection, where transcription occurs several hours after the addition of nucleic acids, microinjection of DNA or mRNA allows for rapid expression and allows for the generation of precise kinetic data.
Cellular Biology, Issue 46, mRNA nuclear export, microinjection, microscopy, fluorescent in situ hybridization, cell biology
RNA-seq Analysis of Transcriptomes in Thrombin-treated and Control Human Pulmonary Microvascular Endothelial Cells
Institutions: Children's Mercy Hospital and Clinics, School of Medicine, University of Missouri-Kansas City.
The characterization of gene expression in cells via measurement of mRNA levels is a useful tool in determining how the transcriptional machinery of the cell is affected by external signals (e.g.
drug treatment), or how cells differ between a healthy state and a diseased state. With the advent and continuous refinement of next-generation DNA sequencing technology, RNA-sequencing (RNA-seq) has become an increasingly popular method of transcriptome analysis to catalog all species of transcripts, to determine the transcriptional structure of all expressed genes and to quantify the changing expression levels of the total set of transcripts in a given cell, tissue or organism1,2
. RNA-seq is gradually replacing DNA microarrays as a preferred method for transcriptome analysis because it has the advantages of profiling a complete transcriptome, providing a digital type datum (copy number of any transcript) and not relying on any known genomic sequence3
Here, we present a complete and detailed protocol to apply RNA-seq to profile transcriptomes in human pulmonary microvascular endothelial cells with or without thrombin treatment. This protocol is based on our recent published study entitled "RNA-seq Reveals Novel Transcriptome of Genes and Their Isoforms in Human Pulmonary Microvascular Endothelial Cells Treated with Thrombin,"4
in which we successfully performed the first complete transcriptome analysis of human pulmonary microvascular endothelial cells treated with thrombin using RNA-seq. It yielded unprecedented resources for further experimentation to gain insights into molecular mechanisms underlying thrombin-mediated endothelial dysfunction in the pathogenesis of inflammatory conditions, cancer, diabetes, and coronary heart disease, and provides potential new leads for therapeutic targets to those diseases.
The descriptive text of this protocol is divided into four parts. The first part describes the treatment of human pulmonary microvascular endothelial cells with thrombin and RNA isolation, quality analysis and quantification. The second part describes library construction and sequencing. The third part describes the data analysis. The fourth part describes an RT-PCR validation assay. Representative results of several key steps are displayed. Useful tips or precautions to boost success in key steps are provided in the Discussion section. Although this protocol uses human pulmonary microvascular endothelial cells treated with thrombin, it can be generalized to profile transcriptomes in both mammalian and non-mammalian cells and in tissues treated with different stimuli or inhibitors, or to compare transcriptomes in cells or tissues between a healthy state and a disease state.
Genetics, Issue 72, Molecular Biology, Immunology, Medicine, Genomics, Proteins, RNA-seq, Next Generation DNA Sequencing, Transcriptome, Transcription, Thrombin, Endothelial cells, high-throughput, DNA, genomic DNA, RT-PCR, PCR
In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection
Institutions: New England Biolabs.
transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A
). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro
transcription has been reported for RNA structure and function studies such as splicing1
, RNAi experiments in mammalian cells2
, antisense RNA amplification by the "Eberwine method"3
, microarray analysis4
and for RNA vaccine studies5
. The technique can also be used for producing radiolabeled and dye labeled probes6
. Warren, et al.
recently reported reprogramming of human cells by transfection with in vitro
transcribed capped RNA7
. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 μg RNA per 20 μl reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8
, efficient translation9
, nuclear transport10
. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia
virus capping enzyme12,13
. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14
such as generating viral genomic RNA for reverse-genetic systems15
and crystallographic studies of cap binding proteins such as eIF4E16
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia
luciferase (GLuc) and Cypridina
luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.
Genetics, Issue 61, In vitro transcription, Vaccinia capping enzyme, transfection, T7 RNA Polymerase, RNA synthesis
Measuring the Kinetics of mRNA Transcription in Single Living Cells
Institutions: Bar-Ilan University.
The transcriptional activity of RNA polymerase II (Pol II) is a dynamic process and therefore measuring the kinetics of the transcriptional process in vivo
is of importance. Pol II kinetics have been measured using biochemical or molecular methods.1-3
In recent years, with the development of new visualization methods, it has become possible to follow transcription as it occurs in real time in single living cells.4
Herein we describe how to perform analysis of Pol II elongation kinetics on a specific gene in living cells.5, 6
Using a cell line in which a specific gene locus (DNA), its mRNA product, and the final protein product can be fluorescently labeled and visualized in vivo
, it is possible to detect the actual transcription of mRNAs on the gene of interest.7, 8
The mRNA is fluorescently tagged using the MS2 system for tagging mRNAs in vivo
, where the 3'UTR of the mRNA transcripts contain 24 MS2 stem-loop repeats, which provide highly specific binding sites for the YFP-MS2 coat protein that labels the mRNA as it is transcribed.9
To monitor the kinetics of transcription we use the Fluorescence Recovery After Photobleaching (FRAP) method. By photobleaching the YFP-MS2-tagged nascent transcripts at the site of transcription and then following the recovery of this signal over time, we obtain the synthesis rate of the newly made mRNAs.5
In other words, YFP-MS2 fluorescence recovery reflects the generation of new MS2 stem-loops in the nascent transcripts and their binding by fluorescent free YFP-MS2 molecules entering from the surrounding nucleoplasm. The FRAP recovery curves are then analyzed using mathematical mechanistic models formalized by a series of differential equations, in order to retrieve the kinetic time parameters of transcription.
Cell Biology, Issue 54, mRNA transcription, nucleus, live-cell imaging, cellular dynamics, FRAP
High-throughput Yeast Plasmid Overexpression Screen
Institutions: University of Pennsylvania School of Medicine , University of Pennsylvania School of Medicine .
The budding yeast, Saccharomyces cerevisiae
, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology 1-5
. In the last few years, several genomewide libraries of yeast strains and plasmids have been generated 6-10
. These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches 11-16
. Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.
Cell Biology, Issue 53, Yeast, plasmid, transformation, PEG/LioAc, high-throughput screen
Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells
Institutions: University of Houston.
Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ERα, and ERβ. ERα is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ERα's regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ERα-positive breast cancer cell lines were compared before and after estrogen-activation using both the µParaflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations, and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.
Medicine, Issue 84, breast cancer, microRNA, estrogen, estrogen receptor, microarray, qPCR
Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System
Institutions: Washington University in St. Louis School of Medicine.
Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1
as well as shift isoform ratios of specific genes 2
. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.
Neurobiology, Issue 75, Neuroscience, Medicine, Biomedical Engineering, Genetics, Anatomy, Physiology, Surgery, Pharmacology, Cerebrospinal Fluid, Rodentia, Oligonucleotides, Antisense, Drug Administration Routes, Injections, Intraventricular, Drug Delivery Systems, mouse, rat, brain, antisense oligonucleotide, osmotic pump, Bolus, Ventricle, Neurosciences, Translational, Cerebrospinal fluid, CNS, cannula, catheter, animal model, surgical techniques
Genetic Manipulation in Δku80 Strains for Functional Genomic Analysis of Toxoplasma gondii
Institutions: The Geisel School of Medicine at Dartmouth.
Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene function and phenotype(s). The development of mutant strains with targeted gene deletions, targeted mutations, complemented gene function, and/or tagged genes provides powerful strategies to address gene function, particularly if these genetic manipulations can be efficiently targeted to the gene locus of interest using integration mediated by double cross over homologous recombination.
Due to very high rates of nonhomologous recombination, functional genomic analysis of Toxoplasma gondii
has been previously limited by the absence of efficient methods for targeting gene deletions and gene replacements to specific genetic loci. Recently, we abolished the major pathway of nonhomologous recombination in type I and type II strains of T. gondii
by deleting the gene encoding the KU80 protein1,2
. The Δku80
strains behave normally during tachyzoite (acute) and bradyzoite (chronic) stages in vitro
and in vivo
and exhibit essentially a 100% frequency of homologous recombination. The Δku80
strains make functional genomic studies feasible on the single gene as well as on the genome scale1-4
Here, we report methods for using type I and type II Δku80Δhxgprt
strains to advance gene targeting approaches in T. gondii
. We outline efficient methods for generating gene deletions, gene replacements, and tagged genes by targeted insertion or deletion of the hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT
) selectable marker. The described gene targeting protocol can be used in a variety of ways in Δku80
strains to advance functional analysis of the parasite genome and to develop single strains that carry multiple targeted genetic manipulations. The application of this genetic method and subsequent phenotypic assays will reveal fundamental and unique aspects of the biology of T. gondii
and related significant human pathogens that cause malaria (Plasmodium
sp.) and cryptosporidiosis (Cryptosporidium
Infectious Diseases, Issue 77, Genetics, Microbiology, Infection, Medicine, Immunology, Molecular Biology, Cellular Biology, Biomedical Engineering, Bioengineering, Genomics, Parasitology, Pathology, Apicomplexa, Coccidia, Toxoplasma, Genetic Techniques, Gene Targeting, Eukaryota, Toxoplasma gondii, genetic manipulation, gene targeting, gene deletion, gene replacement, gene tagging, homologous recombination, DNA, sequencing
Purification of Transcripts and Metabolites from Drosophila Heads
Institutions: University of Florida , University of Florida , University of Florida , University of Florida .
For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila
as a model organism. Although fruit flies provide obvious experimental advantages, research on neurodegenerative diseases has mostly relied on traditional techniques, including genetic interaction, histology, immunofluorescence, and protein biochemistry. These techniques are effective for mechanistic, hypothesis-driven studies, which lead to a detailed understanding of the role of single genes in well-defined biological problems. However, neurodegenerative diseases are highly complex and affect multiple cellular organelles and processes over time. The advent of new technologies and the omics age provides a unique opportunity to understand the global cellular perturbations underlying complex diseases. Flexible model organisms such as Drosophila
are ideal for adapting these new technologies because of their strong annotation and high tractability. One challenge with these small animals, though, is the purification of enough informational molecules (DNA, mRNA, protein, metabolites) from highly relevant tissues such as fly brains. Other challenges consist of collecting large numbers of flies for experimental replicates (critical for statistical robustness) and developing consistent procedures for the purification of high-quality biological material. Here, we describe the procedures for collecting thousands of fly heads and the extraction of transcripts and metabolites to understand how global changes in gene expression and metabolism contribute to neurodegenerative diseases. These procedures are easily scalable and can be applied to the study of proteomic and epigenomic contributions to disease.
Genetics, Issue 73, Biochemistry, Molecular Biology, Neurobiology, Neuroscience, Bioengineering, Cellular Biology, Anatomy, Neurodegenerative Diseases, Biological Assay, Drosophila, fruit fly, head separation, purification, mRNA, RNA, cDNA, DNA, transcripts, metabolites, replicates, SCA3, neurodegeneration, NMR, gene expression, animal model
In Vivo Modeling of the Morbid Human Genome using Danio rerio
Institutions: Duke University Medical Center, Duke University, Duke University Medical Center.
Here, we present methods for the development of assays to query potentially clinically significant nonsynonymous changes using in vivo
complementation in zebrafish. Zebrafish (Danio rerio
) are a useful animal system due to their experimental tractability; embryos are transparent to enable facile viewing, undergo rapid development ex vivo,
and can be genetically manipulated.1
These aspects have allowed for significant advances in the analysis of embryogenesis, molecular processes, and morphogenetic signaling. Taken together, the advantages of this vertebrate model make zebrafish highly amenable to modeling the developmental defects in pediatric disease, and in some cases, adult-onset disorders. Because the zebrafish genome is highly conserved with that of humans (~70% orthologous), it is possible to recapitulate human disease states in zebrafish. This is accomplished either through the injection of mutant human mRNA to induce dominant negative or gain of function alleles, or utilization of morpholino (MO) antisense oligonucleotides to suppress genes to mimic loss of function variants. Through complementation of MO-induced phenotypes with capped human mRNA, our approach enables the interpretation of the deleterious effect of mutations on human protein sequence based on the ability of mutant mRNA to rescue a measurable, physiologically relevant phenotype. Modeling of the human disease alleles occurs through microinjection of zebrafish embryos with MO and/or human mRNA at the 1-4 cell stage, and phenotyping up to seven days post fertilization (dpf). This general strategy can be extended to a wide range of disease phenotypes, as demonstrated in the following protocol. We present our established models for morphogenetic signaling, craniofacial, cardiac, vascular integrity, renal function, and skeletal muscle disorder phenotypes, as well as others.
Molecular Biology, Issue 78, Genetics, Biomedical Engineering, Medicine, Developmental Biology, Biochemistry, Anatomy, Physiology, Bioengineering, Genomics, Medical, zebrafish, in vivo, morpholino, human disease modeling, transcription, PCR, mRNA, DNA, Danio rerio, animal model
Synthesis of an Intein-mediated Artificial Protein Hydrogel
Institutions: Texas A&M University, College Station, Texas A&M University, College Station.
We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans
-splicing reaction. The building blocks of this hydrogel are two protein block-copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. A highly hydrophilic random coil is inserted into one of the block-copolymers for water retention. Mixing of the two protein block copolymers triggers an intein trans
-splicing reaction, yielding a polypeptide unit with crosslinkers at either end that rapidly self-assembles into a hydrogel. This hydrogel is very stable under both acidic and basic conditions, at temperatures up to 50 °C, and in organic solvents. The hydrogel rapidly reforms after shear-induced rupture. Incorporation of a "docking station peptide" into the hydrogel building block enables convenient incorporation of "docking protein"-tagged target proteins. The hydrogel is compatible with tissue culture growth media, supports the diffusion of 20 kDa molecules, and enables the immobilization of bioactive globular proteins. The application of the intein-mediated protein hydrogel as an organic-solvent-compatible biocatalyst was demonstrated by encapsulating the horseradish peroxidase enzyme and corroborating its activity.
Bioengineering, Issue 83, split-intein, self-assembly, shear-thinning, enzyme, immobilization, organic synthesis
Metabolic Labeling of Newly Transcribed RNA for High Resolution Gene Expression Profiling of RNA Synthesis, Processing and Decay in Cell Culture
Institutions: Max von Pettenkofer Institute, University of Cambridge, Ludwig-Maximilians-University Munich.
The development of whole-transcriptome microarrays and next-generation sequencing has revolutionized our understanding of the complexity of cellular gene expression. Along with a better understanding of the involved molecular mechanisms, precise measurements of the underlying kinetics have become increasingly important. Here, these powerful methodologies face major limitations due to intrinsic properties of the template samples they study, i.e.
total cellular RNA. In many cases changes in total cellular RNA occur either too slowly or too quickly to represent the underlying molecular events and their kinetics with sufficient resolution. In addition, the contribution of alterations in RNA synthesis, processing, and decay are not readily differentiated.
We recently developed high-resolution gene expression profiling to overcome these limitations. Our approach is based on metabolic labeling of newly transcribed RNA with 4-thiouridine (thus also referred to as 4sU-tagging) followed by rigorous purification of newly transcribed RNA using thiol-specific biotinylation and streptavidin-coated magnetic beads. It is applicable to a broad range of organisms including vertebrates, Drosophila
, and yeast. We successfully applied 4sU-tagging to study real-time kinetics of transcription factor activities, provide precise measurements of RNA half-lives, and obtain novel insights into the kinetics of RNA processing. Finally, computational modeling can be employed to generate an integrated, comprehensive analysis of the underlying molecular mechanisms.
Genetics, Issue 78, Cellular Biology, Molecular Biology, Microbiology, Biochemistry, Eukaryota, Investigative Techniques, Biological Phenomena, Gene expression profiling, RNA synthesis, RNA processing, RNA decay, 4-thiouridine, 4sU-tagging, microarray analysis, RNA-seq, RNA, DNA, PCR, sequencing
Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun
Institutions: Harvard University, Harvard Medical School, Delft University of Technology.
In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately using complex regulatory strategies including differential promoters and/or signal sequences. Metabolic engineers and synthetic biologists interested in targeting enzymes to a particular organelle are faced with a challenge: For a protein that is to be localized to more than one organelle, the engineer must clone the same gene multiple times. This work presents a solution to this strategy: harnessing alternative splicing of mRNA. This technology takes advantage of established chloroplast and peroxisome targeting sequences and combines them into a single mRNA that is alternatively spliced. Some splice variants are sent to the chloroplast, some to the peroxisome, and some to the cytosol. Here the system is designed for multiple-organelle targeting with alternative splicing. In this work, GFP was expected to be expressed in the chloroplast, cytosol, and peroxisome by a series of rationally designed 5’ mRNA tags. These tags have the potential to reduce the amount of cloning required when heterologous genes need to be expressed in multiple subcellular organelles. The constructs were designed in previous work11
, and were cloned using Gibson assembly, a ligation independent cloning method that does not require restriction enzymes. The resultant plasmids were introduced into Nicotiana benthamiana
epidermal leaf cells with a modified Gene Gun protocol. Finally, transformed leaves were observed with confocal microscopy.
Environmental Sciences, Issue 86, Plant Leaves, Synthetic Biology, Plants, Genetically Modified, DNA, Plant, RNA, Gene Targeting, Plant Physiological Processes, Genes, Gene gun, Gibson assembly, Nicotiana benthamiana, Alternative splicing, confocal microscopy, chloroplast, peroxisome
Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer
Institutions: Max Planck Institute for Psycholinguistics, Donders Institute for Brain, Cognition and Behaviour.
Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis
luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.
Cellular Biology, Issue 87, Protein-protein interactions, Bioluminescence Resonance Energy Transfer, Live cell, Transfection, Luciferase, Yellow Fluorescent Protein, Mutations
A Rapid High-throughput Method for Mapping Ribonucleoproteins (RNPs) on Human pre-mRNA
Institutions: Brown University, Brown University.
Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding location often influences function of a splicing factor. However, as with any sampling strategy the chance of identifying an RNA bound to a splicing factor is proportional to its cellular abundance. We have developed a novel in vitro approach for surveying binding specificity on otherwise transient pre-mRNA. This approach utilizes a specifically designed oligonucleotide pool that tiles across introns, exons, splice junctions, or other pre-mRNA. The pool is subjected to some kind of molecular selection. Here, we demonstrate the method by separating the oligonucleotide into a bound and unbound fraction and utilize a two color array strategy to record the enrichment of each oligonucleotide in the bound fraction. The array data generates high-resolution maps with the ability to identify sequence-specific and structural determinates of ribonucleoprotein (RNP) binding on pre-mRNA. A unique advantage to this method is its ability to avoid the sampling bias towards mRNA associated with current IP and SELEX techniques, as the pool is specifically designed and synthesized from pre-mRNA sequence. The flexibility of the oligonucleotide pool is another advantage since the experimenter chooses which regions to study and tile across, tailoring the pool to their individual needs. Using this technique, one can assay the effects of polymorphisms or mutations on binding on a large scale or clone the library into a functional splicing reporter and identify oligonucleotides that are enriched in the included fraction. This novel in vitro high-resolution mapping scheme provides a unique way to study RNP interactions with transient pre-mRNA species, whose low abundance makes them difficult to study with current in vivo techniques.
Cellular Biology, Issue 34, pre-mRNA, splicing factors, tiling array, ribonucleoprotein (RNP), binding maps
Microinjection of Zebrafish Embryos to Analyze Gene Function
Institutions: Harvard Medical School, Children’s Hospital Boston.
One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo. Morpholinos, which are synthetic oligonucleotides with antisense complementarity to target RNAs, can be added to the embryo to reduce the expression of a particular gene product. Conversely, processed mRNA can be added to the embryo to increase levels of a gene product. The vehicle for adding either mRNA or morpholino to an embryo is microinjection. Microinjection is efficient and rapid, allowing for the injection of hundreds of embryos per hour. This video shows all the steps involved in microinjection. Briefly, eggs are collected immediately after being laid and lined up against a microscope slide in a Petri dish. Next, a fine-tipped needle loaded with injection material is connected to a microinjector and an air source, and the microinjector controls are adjusted to produce a desirable injection volume. Finally, the needle is plunged into the embryo's yolk and the morpholino or mRNA is expelled.
Developmental Biology, Issue 25, zebrafish, morpholino, development, microinjection, heart of glass, heg
Detection of Protein Ubiquitination
Institutions: The Sanford Burnham Institute for Medical Research.
Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of three enzymes. They include ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. Unlike to E1 and E2, E3 ubiquitin ligases display substrate specificity. On the other hand, numerous deubiquitylating enzymes have roles in processing polyubiquitinated proteins. Ubiquitination can result in change of protein stability, cellular localization, and biological activity. Mutations of genes involved in the ubiquitination/deubiquitination pathway or altered ubiquitin system function are associated with many different human diseases such as various types of cancer, neurodegeneration, and metabolic disorders. The detection of altered or normal ubiquitination of target proteins may provide a better understanding on the pathogenesis of these diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo
and test tubes in vitro
. These protocols are also useful to detect other ubiquitin-like small molecule modification such as sumolyation and neddylation.
Cell Biology, Biochemistry, Issue 30, ubiquitination, cultured cell, in vitro system, immunoprecipitation, immunoblotting, ubiquitin, posttranslational modification
A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia
Institutions: Universite de Montreal, Universite de Montreal, Universite de Montreal.
There are several lines of evidence supporting the role of de novo
mutations as a mechanism for common disorders, such as autism and schizophrenia. First, the de novo
mutation rate in humans is relatively high, so new mutations are generated at a high frequency in the population. However, de novo
mutations have not been reported in most common diseases. Mutations in genes leading to severe diseases where there is a strong negative selection against the phenotype, such as lethality in embryonic stages or reduced reproductive fitness, will not be transmitted to multiple family members, and therefore will not be detected by linkage gene mapping or association studies. The observation of very high concordance in monozygotic twins and very low concordance in dizygotic twins also strongly supports the hypothesis that a significant fraction of cases may result from new mutations. Such is the case for diseases such as autism and schizophrenia. Second, despite reduced reproductive fitness1
and extremely variable environmental factors, the incidence of some diseases is maintained worldwide at a relatively high and constant rate. This is the case for autism and schizophrenia, with an incidence of approximately 1% worldwide. Mutational load can be thought of as a balance between selection for or against a deleterious mutation and its production by de novo
mutation. Lower rates of reproduction constitute a negative selection factor that should reduce the number of mutant alleles in the population, ultimately leading to decreased disease prevalence. These selective pressures tend to be of different intensity in different environments. Nonetheless, these severe mental disorders have been maintained at a constant relatively high prevalence in the worldwide population across a wide range of cultures and countries despite a strong negative selection against them2
. This is not what one would predict in diseases with reduced reproductive fitness, unless there was a high new mutation rate. Finally, the effects of paternal age: there is a significantly increased risk of the disease with increasing paternal age, which could result from the age related increase in paternal de novo
mutations. This is the case for autism and schizophrenia3
. The male-to-female ratio of mutation rate is estimated at about 4–6:1, presumably due to a higher number of germ-cell divisions with age in males. Therefore, one would predict that de novo
mutations would more frequently come from males, particularly older males4
. A high rate of new mutations may in part explain why genetic studies have so far failed to identify many genes predisposing to complexes diseases genes, such as autism and schizophrenia, and why diseases have been identified for a mere 3% of genes in the human genome. Identification for de novo
mutations as a cause of a disease requires a targeted molecular approach, which includes studying parents and affected subjects. The process for determining if the genetic basis of a disease may result in part from de novo
mutations and the molecular approach to establish this link will be illustrated, using autism and schizophrenia as examples.
Medicine, Issue 52, de novo mutation, complex diseases, schizophrenia, autism, rare variations, DNA sequencing
A Simple Composite Phenotype Scoring System for Evaluating Mouse Models of Cerebellar Ataxia
Institutions: University of Washington, University of Washington, University of California, San Diego - Rady Children’s Hospital.
We describe a protocol for the rapid and sensitive quantification of disease severity in mouse models of cerebella ataxia. It is derived from previously published phenotype assessments in several disease models, including spinocerebellar ataxias, Huntington s disease and spinobulbar muscular atrophy. Measures include hind limb clasping, ledge test, gait and kyphosis. Each measure is recorded on a scale of 0-3, with a combined total of 0-12 for all four measures. The results effectively discriminate between affected and non-affected individuals, while also quantifying the temporal progression of neurodegenerative disease phenotypes. Measures may be analyzed individually or combined into a composite phenotype score for greater statistical power. The ideal combination of the four described measures will depend upon the disorder in question. We present an example of the protocol used to assess disease severity in a transgenic mouse model of spinocerebellar ataxia type 7 (SCA7).
Albert R. La Spada and Gwenn A. Garden contributed to this manuscript equally.
JoVE Neuroscience, Issue 39, Neurodegeneration, Mouse behavior assay, cerebellar ataxia, polyglutamine disease