Transient kinetic analysis is indispensable for understanding the workings of biological macromolecules, since this approach yields mechanistic information including active site concentrations and intrinsic rate constants that govern macromolecular function. In case of enzymes, for example, transient or pre-steady state measurements identify and characterize individual events in the reaction pathway, whereas steady state measurements only yield overall catalytic efficiency and specificity. Individual events such as protein-protein or protein-ligand interactions and rate-limiting conformational changes often occur in the millisecond timescale, and can be measured directly by stopped-flow and chemical-quench flow methods. Given an optical signal such as fluorescence, stopped-flow serves as a powerful and accessible tool for monitoring reaction progress from substrate binding to product release and catalytic turnover1,2.
Here, we report application of stopped-flow kinetics to probe the mechanism of action of Msh2-Msh6, a eukaryotic DNA repair protein that recognizes base-pair mismatches and insertion/deletion loops in DNA and signals mismatch repair (MMR)3-5. In doing so, Msh2-Msh6 increases the accuracy of DNA replication by three orders of magnitude (error frequency decreases from ~10-6 to10-9 bases), and thus helps preserve genomic integrity. Not surprisingly, defective human Msh2-Msh6 function is associated with hereditary non-polyposis colon cancer and other sporadic cancers6-8. In order to understand the mechanism of action of this critical DNA metabolic protein, we are probing the dynamics of Msh2-Msh6 interaction with mismatched DNA as well as the ATPase activity that fuels its actions in MMR. DNA binding is measured by rapidly mixing Msh2-Msh6 with DNA containing a 2-aminopurine (2-Ap) fluorophore adjacent to a G:T mismatch and monitoring the resulting increase in 2-aminopurine fluorescence in real time. DNA dissociation is measured by mixing pre-formed Msh2-Msh6 G:T(2-Ap) mismatch complex with unlabeled trap DNA and monitoring decrease in fluorescence over time9. Pre-steady state ATPase kinetics are measured by the change in fluorescence of 7-diethylamino-3-((((2-maleimidyl)ethyl)amino)carbonyl) coumarin)-labeled Phosphate Binding Protein (MDCC-PBP) on binding phosphate (Pi) released by Msh2-Msh6 following ATP hydrolysis9,10.
The data reveal rapid binding of Msh2-Msh6 to a G:T mismatch and formation of a long-lived Msh2-Msh6 G:T complex, which in turn results in suppression of ATP hydrolysis and stabilization of the protein in an ATP-bound form. The reaction kinetics provide clear support for the hypothesis that ATP-bound Msh2-Msh6 signals DNA repair on binding a mismatched base pair in the double helix.
F. Noah Biro and Jie Zhai contributed to this paper equally.
22 Related JoVE Articles!
Non-invasive Assessment of the Efficacy of New Therapeutics for Intestinal Pathologies Using Serial Endoscopic Imaging of Live Mice
Institutions: The Walter and Eliza Hall Institute for Medical Research, University of Melbourne, Olivia Newton-John Cancer Research Institute.
Animal models of inflammatory bowel disease (IBD) and colorectal cancer (CRC) have provided significant insight into the cell intrinsic and extrinsic mechanisms that contribute to the onset and progression of intestinal diseases. The identification of new molecules that promote these pathologies has led to a flurry of activity focused on the development of potential new therapies to inhibit their function. As a result, various pre-clinical mouse models with an intact immune system and stromal microenvironment are now heavily used. Here we describe three experimental protocols to test the efficacy of new therapeutics in pre-clinical models of (1) acute mucosal damage, (2) chronic colitis and/or colitis-associated colon cancer, and (3) sporadic colorectal cancer. We also outline procedures for serial endoscopic examination that can be used to document the therapeutic response of an individual tumor and to monitor the health of individual mice. These protocols provide complementary experimental platforms to test the effectiveness of therapeutic compounds shown to be well tolerated by mice.
Medicine, Issue 97, cancer, colitis, colon, endoscopy, mucosa, therapy.
Adaptation of Semiautomated Circulating Tumor Cell (CTC) Assays for Clinical and Preclinical Research Applications
Institutions: London Health Sciences Centre, Western University, London Health Sciences Centre, Lawson Health Research Institute, Western University.
The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the presence of circulating tumor cells (CTCs). These rare cells have been demonstrated to be of clinical significance in metastatic breast, prostate, and colorectal cancers. The current gold standard in clinical CTC detection and enumeration is the FDA-cleared CellSearch system (CSS). This manuscript outlines the standard protocol utilized by this platform as well as two additional adapted protocols that describe the detailed process of user-defined marker optimization for protein characterization of patient CTCs and a comparable protocol for CTC capture in very low volumes of blood, using standard CSS reagents, for studying in vivo
preclinical mouse models of metastasis. In addition, differences in CTC quality between healthy donor blood spiked with cells from tissue culture versus patient blood samples are highlighted. Finally, several commonly discrepant items that can lead to CTC misclassification errors are outlined. Taken together, these protocols will provide a useful resource for users of this platform interested in preclinical and clinical research pertaining to metastasis and CTCs.
Medicine, Issue 84, Metastasis, circulating tumor cells (CTCs), CellSearch system, user defined marker characterization, in vivo, preclinical mouse model, clinical research
Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts
Institutions: University of Southampton School of Medicine, University of Southampton School of Medicine, London Research Institute, Cancer Research UK.
Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the extracellular matrix (ECM). Characterizing the biology of this phenotypically distinct group of cells could substantially improve our understanding of early events during the metastatic cascade.
Tumor invasion is a dynamic process facilitated by bidirectional interactions between malignant epithelium and the cancer associated stroma. In order to examine cell-specific responses at the tumor stroma-interface we have combined organotypic co-culture and laser micro-dissection techniques.
Organotypic models, in which key stromal constituents such as fibroblasts are 3-dimentioanally co-cultured with cancer epithelial cells, are highly manipulatable experimental tools which enable invasion and cancer-stroma interactions to be studied in near-physiological conditions.
Laser microdissection (LMD) is a technique which entails the surgical dissection and extraction of the various strata within tumor tissue, with micron level precision.
By combining these techniques with genomic, transcriptomic and epigenetic profiling we aim to develop a deeper understanding of the molecular characteristics of invading tumor cells and surrounding stromal tissue, and in doing so potentially reveal novel biomarkers and opportunities for drug development in CRC.
Medicine, Issue 86, Colorectal cancer, Cancer metastasis, organotypic culture, laser microdissection, molecular profiling, invasion, tumor microenvironment, stromal tissue, epithelium, fibroblasts
Transgenic Rodent Assay for Quantifying Male Germ Cell Mutant Frequency
Institutions: Environmental Health Centre.
mutations arise mostly in the male germline and may contribute to adverse health outcomes in subsequent generations. Traditional methods for assessing the induction of germ cell mutations require the use of large numbers of animals, making them impractical. As such, germ cell mutagenicity is rarely assessed during chemical testing and risk assessment. Herein, we describe an in vivo
male germ cell mutation assay using a transgenic rodent model that is based on a recently approved Organisation for Economic Co-operation and Development (OECD) test guideline. This method uses an in vitro
positive selection assay to measure in vivo
mutations induced in a transgenic λgt10 vector bearing a reporter gene directly in the germ cells of exposed males. We further describe how the detection of mutations in the transgene recovered from germ cells can be used to characterize the stage-specific sensitivity of the various spermatogenic cell types to mutagen exposure by controlling three experimental parameters: the duration of exposure (administration time), the time between exposure and sample collection (sampling time), and the cell population collected for analysis. Because a large number of germ cells can be assayed from a single male, this method has superior sensitivity compared with traditional methods, requires fewer animals and therefore much less time and resources.
Genetics, Issue 90, sperm, spermatogonia, male germ cells, spermatogenesis, de novo mutation, OECD TG 488, transgenic rodent mutation assay, N-ethyl-N-nitrosourea, genetic toxicology
Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice
Institutions: University of North Carolina School of Medicine, University of North Carolina School of Medicine, University of North Carolina School of Medicine, University of North Carolina School of Medicine, University of North Carolina School of Medicine, Emory University School of Medicine, University of North Carolina School of Medicine.
Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro
using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro
. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo
. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo
tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro
and in vivo
and may be useful in preclinical drug development for these devastating diseases.
Neuroscience, Issue 90, astrocytoma, cortical astrocytes, genetically engineered mice, glioblastoma, neural stem cells, orthotopic allograft
Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation
Institutions: University Hospital Münster, University Children's Hospital Münster.
Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions preclinically. However, valid assessment of pathological alterations often requires histological analysis, and when performed ex vivo,
necessitates death of the animal. Therefore in conventional experimental settings, intra-individual follow-up examinations are rarely possible. Thus, development of murine endoscopy in live
mice enables investigators for the first time to both directly visualize the gastrointestinal mucosa and also repeat the procedure to monitor for alterations. Numerous applications for in vivo
murine endoscopy exist, including studying intestinal inflammation or wound healing, obtaining mucosal biopsies repeatedly, and to locally administer diagnostic or therapeutic agents using miniature injection catheters. Most recently, molecular imaging has extended diagnostic imaging modalities allowing specific detection of distinct target molecules using specific photoprobes. In conclusion, murine endoscopy has emerged as a novel cutting-edge technology for diagnostic experimental in vivo
imaging and may significantly impact on preclinical research in various fields.
Medicine, Issue 90,
gastroenterology, in vivo imaging, murine endoscopy, diagnostic imaging, carcinogenesis, intestinal wound healing, experimental colitis
A Next-generation Tissue Microarray (ngTMA) Protocol for Biomarker Studies
Institutions: University of Bern.
Biomarker research relies on tissue microarrays (TMA). TMAs are produced by repeated transfer of small tissue cores from a ‘donor’ block into a ‘recipient’ block and then used for a variety of biomarker applications. The construction of conventional TMAs is labor intensive, imprecise, and time-consuming. Here, a protocol using next-generation Tissue Microarrays (ngTMA) is outlined. ngTMA is based on TMA planning and design, digital pathology, and automated tissue microarraying. The protocol is illustrated using an example of 134 metastatic colorectal cancer patients. Histological, statistical and logistical aspects are considered, such as the tissue type, specific histological regions, and cell types for inclusion in the TMA, the number of tissue spots, sample size, statistical analysis, and number of TMA copies. Histological slides for each patient are scanned and uploaded onto a web-based digital platform. There, they are viewed and annotated (marked) using a 0.6-2.0 mm diameter tool, multiple times using various colors to distinguish tissue areas. Donor blocks and 12 ‘recipient’ blocks are loaded into the instrument. Digital slides are retrieved and matched to donor block images. Repeated arraying of annotated regions is automatically performed resulting in an ngTMA. In this example, six ngTMAs are planned containing six different tissue types/histological zones. Two copies of the ngTMAs are desired. Three to four slides for each patient are scanned; 3 scan runs are necessary and performed overnight. All slides are annotated; different colors are used to represent the different tissues/zones, namely tumor center, invasion front, tumor/stroma, lymph node metastases, liver metastases, and normal tissue. 17 annotations/case are made; time for annotation is 2-3 min/case. 12 ngTMAs are produced containing 4,556 spots. Arraying time is 15-20 hr. Due to its precision, flexibility and speed, ngTMA is a powerful tool to further improve the quality of TMAs used in clinical and translational research.
Medicine, Issue 91, tissue microarray, biomarkers, prognostic, predictive, digital pathology, slide scanning
gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair
Institutions: Centre Georges-François Leclerc.
The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on cancer, and especially cancer genetics. Current knowledge and future discoveries will make it necessary to study a huge number of genes that could be involved in a genetic predisposition to cancer. In this regard, we developed a Nextera design to study 11 complete genes involved in DNA damage repair. This protocol was developed to safely study 11 genes (ATM
, and TP53
) from promoter to 3'-UTR in 24 patients simultaneously. This protocol, based on transposase technology and gDNA enrichment, gives a great advantage in terms of time for the genetic diagnosis thanks to sample multiplexing. This protocol can be safely used with blood gDNA.
Genetics, Issue 92, gDNA enrichment, Nextera, NGS, DNA damage, BRCA1, BRCA2
In vitro Functional Characterization of Mouse Colorectal Afferent Endings
Institutions: University of Pittsburgh.
This video demonstrates in detail an in vitro
single-fiber electrophysiological recording protocol using a mouse colorectum-nerve preparation. The approach allows unbiased identification and functional characterization of individual colorectal afferents. Extracellular recordings of propagated action potentials (APs) that originate from one or a few afferent (i.e.,
single-fiber) receptive fields (RFs) in the colorectum are made from teased nerve fiber fascicles. The colorectum is removed with either the pelvic (PN) or lumbar splanchnic (LSN) nerve attached and opened longitudinally. The tissue is placed in a recording chamber, pinned flat and perfused with oxygenated Krebs solution. Focal electrical stimulation is used to locate the colorectal afferent endings, which are further tested by three distinct mechanical stimuli (blunt probing, mucosal stroking and circumferential stretch) to functionally categorize the afferents into five mechanosensitive classes. Endings responding to none of these mechanical stimuli are categorized as mechanically-insensitive afferents (MIAs). Both mechanosensitive and MIAs can be assessed for sensitization (i.e.,
enhanced response, reduced threshold, and/or acquisition of mechanosensitivity) by localized exposure of RFs to chemicals (e.g.,
inflammatory soup (IS), capsaicin, adenosine triphosphate (ATP)). We describe the equipment and colorectum–nerve recording preparation, harvest of colorectum with attached PN or LSN, identification of RFs in the colorectum, single-fiber recording from nerve fascicles, and localized application of chemicals to the RF. In addition, challenges of the preparation and application of standardized mechanical stimulation are also discussed.
Medicine, Issue 95, visceral afferent, colorectum, single-fiber, extracellular recording, teased fiber, pelvic, lumbar splanchnic, mechanically-insensitive afferent, MIA, silent afferent
Flexible Colonoscopy in Mice to Evaluate the Severity of Colitis and Colorectal Tumors Using a Validated Endoscopic Scoring System
Institutions: Case Western Reserve University School of Medicine, Cleveland, Case Western Reserve University School of Medicine, Cleveland, Case Western Reserve University School of Medicine, Cleveland.
The use of modern endoscopy for research purposes has greatly facilitated our understanding of gastrointestinal pathologies. In particular, experimental endoscopy has been highly useful for studies that require repeated assessments in a single laboratory animal, such as those evaluating mechanisms of chronic inflammatory bowel disease and the progression of colorectal cancer. However, the methods used across studies are highly variable. At least three endoscopic scoring systems have been published for murine colitis and published protocols for the assessment of colorectal tumors fail to address the presence of concomitant colonic inflammation. This study develops and validates a reproducible endoscopic scoring system that integrates evaluation of both inflammation and tumors simultaneously. This novel scoring system has three major components: 1) assessment of the extent and severity of colorectal inflammation (based on perianal findings, transparency of the wall, mucosal bleeding, and focal lesions), 2) quantitative recording of tumor lesions (grid map and bar graph), and 3) numerical sorting of clinical cases by their pathological and research relevance based on decimal units with assigned categories of observed lesions and endoscopic complications (decimal identifiers). The video and manuscript presented herein were prepared, following IACUC-approved protocols, to allow investigators to score their own experimental mice using a well-validated and highly reproducible endoscopic methodology, with the system option to differentiate distal from proximal endoscopic colitis (D-PECS).
Medicine, Issue 80, Crohn's disease, ulcerative colitis, colon cancer, Clostridium difficile, SAMP mice, DSS/AOM-colitis, decimal scoring identifier
Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells
Institutions: UT Health Science Center at San Antonio, UT Health Science Center at San Antonio, UT Health Science Center at San Antonio, UT Health Science Center at San Antonio, UT Health Science Center at San Antonio.
In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem cells are responsible for ensuring tissue homeostasis and repair throughout life, it is reasonable to hypothesize that the stem cell population is critical for preserving genomic integrity of tissues. Therefore, significant interest has arisen in assessing the impact of endogenous and environmental factors on genomic integrity in stem cells and their progeny, aiming to understand the etiology of stem-cell based diseases.
transgenic mice carry a recoverable λ phage vector encoding the LacI
reporter system, in which the LacI
gene serves as the mutation reporter. The result of a mutated LacI
gene is the production of β-galactosidase that cleaves a chromogenic substrate, turning it blue. The LacI
reporter system is carried in all cells, including stem/progenitor cells and can easily be recovered and used to subsequently infect E. coli
. After incubating infected E. coli
on agarose that contains the correct substrate, plaques can be scored; blue plaques indicate a mutant LacI
gene, while clear plaques harbor wild-type. The frequency of blue (among clear) plaques indicates the mutant frequency in the original cell population the DNA was extracted from. Sequencing the mutant LacI
gene will show the location of the mutations in the gene and the type of mutation.
transgenic mouse model is well-established as an in vivo
mutagenesis assay. Moreover, the mice and the reagents for the assay are commercially available. Here we describe in detail how this model can be adapted to measure the frequency of spontaneously occurring DNA mutants in stem cell-enriched Lin-
(LSK) cells and other subpopulations of the hematopoietic system.
Infection, Issue 84, In vivo mutagenesis, hematopoietic stem/progenitor cells, LacI mouse model, DNA mutations, E. coli
Detecting Somatic Genetic Alterations in Tumor Specimens by Exon Capture and Massively Parallel Sequencing
Institutions: Memorial Sloan-Kettering Cancer Center, Memorial Sloan-Kettering Cancer Center.
Efforts to detect and investigate key oncogenic mutations have proven valuable to facilitate the appropriate treatment for cancer patients. The establishment of high-throughput, massively parallel "next-generation" sequencing has aided the discovery of many such mutations. To enhance the clinical and translational utility of this technology, platforms must be high-throughput, cost-effective, and compatible with formalin-fixed paraffin embedded (FFPE) tissue samples that may yield small amounts of degraded or damaged DNA. Here, we describe the preparation of barcoded and multiplexed DNA libraries followed by hybridization-based capture of targeted exons for the detection of cancer-associated mutations in fresh frozen and FFPE tumors by massively parallel sequencing. This method enables the identification of sequence mutations, copy number alterations, and select structural rearrangements involving all targeted genes. Targeted exon sequencing offers the benefits of high throughput, low cost, and deep sequence coverage, thus conferring high sensitivity for detecting low frequency mutations.
Molecular Biology, Issue 80, Molecular Diagnostic Techniques, High-Throughput Nucleotide Sequencing, Genetics, Neoplasms, Diagnosis, Massively parallel sequencing, targeted exon sequencing, hybridization capture, cancer, FFPE, DNA mutations
Generation of Comprehensive Thoracic Oncology Database - Tool for Translational Research
Institutions: University of Chicago, University of Chicago, Northshore University Health Systems, University of Chicago, University of Chicago, University of Chicago.
The Thoracic Oncology Program Database Project was created to serve as a comprehensive, verified, and accessible repository for well-annotated cancer specimens and clinical data to be available to researchers within the Thoracic Oncology Research Program. This database also captures a large volume of genomic and proteomic data obtained from various tumor tissue studies. A team of clinical and basic science researchers, a biostatistician, and a bioinformatics expert was convened to design the database. Variables of interest were clearly defined and their descriptions were written within a standard operating manual to ensure consistency of data annotation. Using a protocol for prospective tissue banking and another protocol for retrospective banking, tumor and normal tissue samples from patients consented to these protocols were collected. Clinical information such as demographics, cancer characterization, and treatment plans for these patients were abstracted and entered into an Access database. Proteomic and genomic data have been included in the database and have been linked to clinical information for patients described within the database. The data from each table were linked using the relationships function in Microsoft Access to allow the database manager to connect clinical and laboratory information during a query. The queried data can then be exported for statistical analysis and hypothesis generation.
Medicine, Issue 47, Database, Thoracic oncology, Bioinformatics, Biorepository, Microsoft Access, Proteomics, Genomics
Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1
Institutions: The Ohio State University, Tohoku University.
The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the BRCA1 have been complicated by the lack of a robust assay for the full length BRCA1 protein and the difficulties inherent in working with cell lines that express hypomorphic BRCA1 protein1,2,3,4,5
. We developed a system whereby the endogenous BRCA1 protein in a cell was acutely depleted by RNAi targeting the 3'-UTR of the BRCA1 mRNA and replaced by co-transfecting a plasmid expressing a BRCA1 variant. One advantage of this procedure is that the acute silencing of BRCA1 and simultaneous replacement allow the cells to grow without secondary mutations or adaptations that might arise over time to compensate for the loss of BRCA1 function. This depletion and add-back procedure was done in a HeLa-derived cell line that was readily assayed for homologous recombination activity. The homologous recombination assay is based on a previously published method whereby a recombination substrate is integrated into the genome (Figure 1)6,7,8,9
. This recombination substrate has the rare-cutting I-SceI restriction enzyme site inside an inactive GFP allele, and downstream is a second inactive GFP allele. Transfection of the plasmid that expresses I-SceI results in a double-stranded break, which may be repaired by homologous recombination, and if homologous recombination does repair the break it creates an active GFP allele that is readily scored by flow cytometry for GFP protein expression. Depletion of endogenous BRCA1 resulted in an 8-10-fold reduction in homologous recombination activity, and add-back of wild-type plasmid fully restored homologous recombination function. When specific point mutants of full length BRCA1 were expressed from co-transfected plasmids, the effect of the specific missense mutant could be scored. As an example, the expression of the BRCA1(M18T) protein, a variant of unknown clinical significance10
, was expressed in these cells, it failed to restore BRCA1-dependent homologous recombination. By contrast, expression of another variant, also of unknown significance, BRCA1(I21V) fully restored BRCA1-dependent homologous recombination function. This strategy of testing the function of BRCA1 missense mutations has been applied to another biological system assaying for centrosome function (Kais et al, unpublished observations). Overall, this approach is suitable for the analysis of missense mutants in any gene that must be analyzed recessively.
Cell Biology, Issue 48, BRCA1, homologous recombination, breast cancer, RNA interference, DNA repair
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
Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing
Institutions: University of Sydney, Royal Prince Alfred Hospital, Department of Anatomical Pathology, Concord Repatriation General Hospital.
The current prognosis and classification of CRC relies on staging systems that integrate histopathologic and clinical findings. However, in the majority of CRC cases, cell dysfunction is the result of numerous mutations that modify protein expression and post-translational modification1
A number of cell surface antigens, including cluster of differentiation (CD) antigens, have been identified as potential prognostic or metastatic biomarkers in CRC. These antigens make ideal biomarkers as their expression often changes with tumour progression or interactions with other cell types, such as tumour-infiltrating lymphocytes (TILs) and tumour-associated macrophages (TAMs).
The use of immunohistochemistry (IHC) for cancer sub-classification and prognostication is well established for some tumour types2,3
. However, no single ‘marker’ has shown prognostic significance greater than clinico-pathological staging or gained wide acceptance for use in routine pathology reporting of all CRC cases.
A more recent approach to prognostic stratification of disease phenotypes relies on surface protein profiles using multiple 'markers'. While expression profiling of tumours using proteomic techniques such as iTRAQ is a powerful tool for the discovery of biomarkers4, it is not optimal for routine use in diagnostic laboratories and cannot distinguish different cell types in a mixed population. In addition, large amounts of tumour tissue are required for the profiling of purified plasma membrane glycoproteins by these methods.
In this video we described a simple method for surface proteome profiling of viable cells from disaggregated CRC samples using a DotScan CRC antibody microarray. The 122-antibody microarray consists of a standard 82-antibody region recognizing a range of lineage-specific leukocyte markers, adhesion molecules, receptors and markers of inflammation and immune response5
, together with a satellite region for detection of 40 potentially prognostic markers for CRC. Cells are captured only on antibodies for which they express the corresponding antigen. The cell density per dot, determined by optical scanning, reflects the proportion of cells expressing that antigen, the level of expression of the antigen and affinity of the antibody6
For CRC tissue or normal intestinal mucosa, optical scans reflect the immunophenotype of mixed populations of cells. Fluorescence multiplexing can then be used to profile selected sub-populations of cells of interest captured on the array. For example, Alexa 647-anti-epithelial cell adhesion molecule (EpCAM; CD326), is a pan-epithelial differentiation antigen that was used to detect CRC cells and also epithelial cells of normal intestinal mucosa, while Phycoerythrin-anti-CD3, was used to detect infiltrating T-cells7
. The DotScan CRC microarray should be the prototype for a diagnostic alternative to the anatomically-based CRC staging system.
Immunology, Issue 55, colorectal cancer, leukocytes, antibody microarray, multiplexing, fluorescence, CD antigens
Assessing Somatic Hypermutation in Ramos B Cells after Overexpression or Knockdown of Specific Genes
Institutions: Duke University .
B cells start their life with low affinity antibodies generated by V(D)J recombination. However, upon detecting a pathogen, the variable (V) region of an immunoglobulin (Ig) gene is mutated approximately 100,000-fold more than the rest of the genome through somatic hypermutation (SHM), resulting in high affinity antibodies1,2
. In addition, class switch recombination (CSR) produces antibodies with different effector functions depending on the kind of immune response that is needed for a particular pathogen. Both CSR and SHM are initiated by activation-induced cytidine deaminase (AID), which deaminates cytosine residues in DNA to produce uracils. These uracils are processed by error-prone forms of repair pathways, eventually leading to mutations and recombination1-3
Our current understanding of the molecular details of SHM and CSR come from a combination of studies in mice, primary cells, cell lines, and cell-free experiments. Mouse models remain the gold standard with genetic knockouts showing critical roles for many repair factors (e.g. Ung, Msh2, Msh6, Exo1, and polymerase η)4-10
. However, not all genes are amenable for knockout studies. For example, knockouts of several double-strand break repair proteins are embryonically lethal or impair B-cell development11-14
. Moreover, sometimes the specific function of a protein in SHM or CSR may be masked by more global defects caused by the knockout. In addition, since experiments in mice can be lengthy, altering expression of individual genes in cell lines has become an increasingly popular first step to identifying and characterizing candidate genes15-18
Ramos – a Burkitt lymphoma cell line that constitutively undergoes SHM – has been a popular cell-line model to study SHM18-24
. One advantage of Ramos cells is that they have a built-in convenient semi-quantitative measure of SHM. Wild type cells express IgM and, as they pick up mutations, some of the mutations knock out IgM expression. Therefore, assaying IgM loss by fluorescence-activated cell scanning (FACS) provides a quick read-out for the level of SHM. A more quantitative measurement of SHM can be obtained by directly sequencing the antibody genes.
Since Ramos cells are difficult to transfect, we produce stable derivatives that have increased or lowered expression of an individual gene by infecting cells with retroviral or lentiviral constructs that contain either an overexpression cassette or a short hairpin RNA (shRNA), respectively. Here, we describe how we infect Ramos cells and then use these cells to investigate the role of specific genes on SHM (Figure 1).
Immunology, Issue 57, activation-induced cytidine deaminase, lentiviral infection, retroviral infection, Ramos, shRNA, somatic hypermutation
Identification of Sleeping Beauty Transposon Insertions in Solid Tumors using Linker-mediated PCR
Institutions: University of Minnesota, Minneapolis, University of Minnesota, Minneapolis.
Genomic, proteomic, transcriptomic, and epigenomic analyses of human tumors indicate that there are thousands of anomalies within each cancer genome compared to matched normal tissue. Based on these analyses it is evident that there are many undiscovered genetic drivers of cancer1
. Unfortunately these drivers are hidden within a much larger number of passenger anomalies in the genome that do not directly contribute to tumor formation. Another aspect of the cancer genome is that there is considerable genetic heterogeneity within similar tumor types. Each tumor can harbor different mutations that provide a selective advantage for tumor formation2
. Performing an unbiased forward genetic screen in mice provides the tools to generate tumors and analyze their genetic composition, while reducing the background of passenger mutations. The Sleeping Beauty
(SB) transposon system is one such method3
. The SB system utilizes mobile vectors (transposons) that can be inserted throughout the genome by the transposase enzyme. Mutations are limited to a specific cell type through the use of a conditional transposase allele that is activated by Cre Recombinase
. Many mouse lines exist that express Cre Recombinase
in specific tissues. By crossing one of these lines to the conditional transposase allele (e.g.
Lox-stop-Lox-SB11), the SB system is activated only in cells that express Cre Recombinase
. The Cre Recombinase
will excise a stop cassette that blocks expression of the transposase allele, thereby activating transposon mutagenesis within the designated cell type. An SB screen is initiated by breeding three strains of transgenic mice so that the experimental mice carry a conditional transposase allele, a concatamer of transposons, and a tissue-specific Cre Recombinase
allele. These mice are allowed to age until tumors form and they become moribund. The mice are then necropsied and genomic DNA is isolated from the tumors. Next, the genomic DNA is subjected to linker-mediated-PCR (LM-PCR) that results in amplification of genomic loci containing an SB transposon. LM-PCR performed on a single tumor will result in hundreds of distinct amplicons representing the hundreds of genomic loci containing transposon insertions in a single tumor4
. The transposon insertions in all tumors are analyzed and common insertion sites (CISs) are identified using an appropriate statistical method5
. Genes within the CIS are highly likely to be oncogenes or tumor suppressor genes, and are considered candidate cancer genes. The advantages of using the SB system to identify candidate cancer genes are: 1) the transposon can easily be located in the genome because its sequence is known, 2) transposition can be directed to almost any cell type and 3) the transposon is capable of introducing both gain- and loss-of-function mutations6
. The following protocol describes how to devise and execute a forward genetic screen using the SB transposon system to identify candidate cancer genes (Figure 1
Genetics, Issue 72, Medicine, Cancer Biology, Biomedical Engineering, Genomics, Mice, Genetic Techniques, life sciences, animal models, Neoplasms, Genetic Phenomena, Forward genetic screen, cancer drivers, mouse models, oncogenes, tumor suppressor genes, Sleeping Beauty transposons, insertions, DNA, PCR, animal model
In vitro Organoid Culture of Primary Mouse Colon Tumors
Institutions: University of Michigan , University of Michigan .
Several human and murine colon cancer cell lines have been established, physiologic integrity of colon tumors such as multiple cell layers, basal-apical polarity, ability to differentiate, and anoikis are not maintained in colon cancer derived cell lines. The present study demonstrates a method for culturing primary mouse colon tumor organoids adapted from Sato T et al. 1
, which retains important physiologic features of colon tumors. This method consists of mouse colon tumor tissue collection, adjacent normal colon epithelium dissociation, colon tumor cells digestion into single cells, embedding colon tumor cells into matrigel, and selective culture based on the principle that tumor cells maintain growth on limiting nutrient conditions compared to normal epithelial cells.
The primary tumor organoids if isolated from genetically modified mice provide a very useful system to assess tumor autonomous function of specific genes. Moreover, the tumor organoids are amenable to genetic manipulation by virus meditated gene delivery; therefore signaling pathways involved in the colon tumorigenesis could also be extensively investigated by overexpression or knockdown. Primary tumor organoids culture provides a physiologic relevant and feasible means to study the mechanisms and therapeutic modalities for colon tumorigenesis.
Cancer Biology, Issue 75, Medicine, Molecular Biology, Cellular Biology, Biomedical Engineering, Anatomy, Physiology, Genetics, Oncology, Surgery, Organoids, Tumor Cells, Cultured Colonic Neoplasms, Primary Cell Culture, Colon tumor, chelation, collagenase, matrigel, organoid, EGF, colon cancer, cancer, tumor, cell, isolation, immunohistochemistry, mouse, animal model
The Use of Reverse Phase Protein Arrays (RPPA) to Explore Protein Expression Variation within Individual Renal Cell Cancers
Institutions: University of Edinburgh, University of St Andrews, University of Edinburgh, University of Edinburgh, Western General Hospital, University of Edinburgh, Queen Mary University of London.
Currently there is no curative treatment for metastatic clear cell renal cell cancer, the commonest variant of the disease. A key factor in this treatment resistance is thought to be the molecular complexity of the disease 1
. Targeted therapy such as the tyrosine kinase inhibitor (TKI)-sunitinib have been utilized, but only 40% of patients will respond, with the overwhelming majority of these patients relapsing within 1 year 2
. As such the question of intrinsic and acquired resistance in renal cell cancer patients is highly relevant 3
In order to study resistance to TKIs, with the ultimate goal of developing effective, personalized treatments, sequential tissue after a specific period of targeted therapy is required, an approach which had proved successful in chronic myeloid leukaemia 4
. However the application of such a strategy in renal cell carcinoma is complicated by the high level of both inter- and intratumoral heterogeneity, which is a feature of renal cell carcinoma5,6
as well as other solid tumors 7
. Intertumoral heterogeneity due to transcriptomic and genetic differences is well established even in patients with similar presentation, stage and grade of tumor. In addition it is clear that there is great morphological (intratumoral) heterogeneity in RCC, which is likely to represent even greater molecular heterogeneity. Detailed mapping and categorization of RCC tumors by combined morphological analysis and Fuhrman grading allows the selection of representative areas for proteomic analysis.
Protein based analysis of RCC8
is attractive due to its widespread availability in pathology laboratories; however, its application can be problematic due to the limited availability of specific antibodies 9
. Due to the dot blot nature of the Reverse Phase Protein Arrays (RPPA), antibody specificity must be pre-validated; as such strict quality control of antibodies used is of paramount importance. Despite this limitation the dot blot format does allow assay miniaturization, allowing for the printing of hundreds of samples onto a single nitrocellulose slide. Printed slides can then be analyzed in a similar fashion to Western analysis with the use of target specific primary antibodies and fluorescently labelled secondary antibodies, allowing for multiplexing. Differential protein expression across all the samples on a slide can then be analyzed simultaneously by comparing the relative level of fluorescence in a more cost-effective and high-throughput manner.
Cancer Biology, Issue 71, Bioengineering, Medicine, Biomedical Engineering, Cellular Biology, Molecular Biology, Genetics, Pathology, Oncology, Proteins, Early Detection of Cancer, Translational Medical Research, RPPA, RCC, Heterogeneity, Proteomics, Tumor Grade, intertumoral, tumor, metastatic, carcinoma, renal cancer, clear cell renal cell cancer, cancer, assay
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
Mosaic Zebrafish Transgenesis for Functional Genomic Analysis of Candidate Cooperative Genes in Tumor Pathogenesis
Institutions: Mayo Clinic College of Medicine, Center for Individualized Medicine, Tufts University School of Medicine, Mayo Clinic.
Comprehensive genomic analysis has uncovered surprisingly large numbers of genetic alterations in various types of cancers. To robustly and efficiently identify oncogenic “drivers” among these tumors and define their complex relationships with concurrent genetic alterations during tumor pathogenesis remains a daunting task. Recently, zebrafish have emerged as an important animal model for studying human diseases, largely because of their ease of maintenance, high fecundity, obvious advantages for in vivo
imaging, high conservation of oncogenes and their molecular pathways, susceptibility to tumorigenesis and, most importantly, the availability of transgenic techniques suitable for use in the fish. Transgenic zebrafish models of cancer have been widely used to dissect oncogenic pathways in diverse tumor types. However, developing a stable transgenic fish model is both tedious and time-consuming, and it is even more difficult and more time-consuming to dissect the cooperation of multiple genes in disease pathogenesis using this approach, which requires the generation of multiple transgenic lines with overexpression of the individual genes of interest followed by complicated breeding of these stable transgenic lines. Hence, use of a mosaic transient transgenic approach in zebrafish offers unique advantages for functional genomic analysis in vivo
. Briefly, candidate transgenes can be coinjected into one-cell-stage wild-type or transgenic zebrafish embryos and allowed to integrate together into each somatic cell in a mosaic pattern that leads to mixed genotypes in the same primarily injected animal. This permits one to investigate in a faster and less expensive manner whether and how the candidate genes can collaborate with each other to drive tumorigenesis. By transient overexpression of activated ALK
in the transgenic fish overexpressing MYCN
, we demonstrate here the cooperation of these two oncogenes in the pathogenesis of a pediatric cancer, neuroblastoma that has resisted most forms of contemporary treatment.
Developmental Biology, Issue 97, zebrafish, animal model, mosaic transgenesis, coinjection, functional genomics, tumor initiation