The Functional Genomics Core (FGC) provides reagents, strategic advice, and technical expertise required for efficient investigation of genetic variants likely to be mechanistically important in pediatric rheumatic and inflammatory diseases, and the experiments supported by this core will uncover fundamental mechanisms of disease pathogenesis with broad scientific and clinical implications. In doing so, the FGC will enhance research productivity and efficiency by effectively reducing the “start-up” time required to implement these complex experimental systems, thus reducing costs and effort required and allowing for better study designs and higher quality studies than if investigators were to individually establish this technology. Further, this core will provide access to state-of-the-art technology without requiring the users to become experts in these technologies. In this way the core becomes a practical vehicle for the translation of basic advances to clinical disease applications and their very important insights in to pathogenesis. We expect the FGC to be a hub of innovation for the research community, connecting scientists with the tools and expertise to take their research projects to the next level and to accelerate the discovery of the molecular mechanisms that are responsible for the genetic associations observed.
Standard Methodologies used by the Functional Genomics Core
- Quantitative real time Polymerase Chain Reactions (RT-qPCRs) for allele-specific gene expression: We will support allele-specific RT-qPCR to confirm differential expression of predicted variants using the same TaqMan SNP genotyping assay described above and present these protocols as examples. Experiments will be done in 96-well plates. Input DNA will be compared to complementary DNA (cDNA) for each sample with the expectation that the allelic ratio of the two fluorescent probes is 50:50 for the DNA and demonstrates an allelic imbalance for the cDNA.\
- EMSAs (electrophoretic mobility shift assays): We will use nuclear lysates from our B cell lines, and fluorescently-labeled double-stranded DNA oligonucleotides (dsDNA oligos or oligos) containing the non-risk or risk alleles. As positive controls, we will use oligos with consensus binding sites for each transcription factor. As a negative control, we will use oligos with mutated binding sites. Unlabeled oligos will be used in a competition assay to demonstrate band specificity. To establish binding of specific transcription factors, we will individually “super shift” the complexes with an antibody against each transcription factor. Non-specific antibodies that are the same isotype as the supershifting antibody (e.g., anti-IgG) will be used as a negative control. Bands are imaged using a fluorescence-detecting gel imager.
- DAPA/Westerns: DAPAs (DNA affinity precipitation assays) are a valuable method that complements the EMSA. This approach complements the EMSA by testing the same hypothesis while exploring binding under reducing gel conditions. We will enhance the chance of identifying differences in binding by using both approaches, as sometimes one of the procedures identifies a protein that is not detected by the other approach. As western blots have much higher sensitivity compared to EMSAs, DAPA/Western also allows for quantification of differential binding through densitometry analysis. We will use non-risk, risk, or control oligos (these oligos will be the same sequence as in the EMSAs but will have biotin labels) to precipitate proteins from nuclear lysate using streptavidin coated magnetic beads. This is a column-based method with elution of the DNA oligo-bound material. The eluted proteins will be electrophoresed through a polyacrylamide gel and the bands will be visualized using a silver stain. Western blots will be performed using an antibody to the predicted transcription factor for detection.
- ChIP-qPCRs: We will confirm differential binding of transcription factors to variant risk and non-risk alleles in the nuclei of our B cell lines. We will select cell lines that are homozygous risk, homozygous non-risk and heterozygous for the variant. We will perform ChIP for the predicted transcription factor and use the immunoprecipitated DNA to assess the presence of the DNA surrounding the risk and non-risk variants. These assays will be facilitated by TaqMan SNP genotyping assays that provide one set of primers and two probes with different fluorescent labels (one for each allele). We will encourage investigators to use at least 3 homozygous samples (3 risk and 3 non-risk) as controls and at least 5 heterozygous samples—an experimental design that gives 80% power to detect 40% changes in binding. Based on the fluorescence measured from each probe, we can calculate 1) if the transcription factor binds the region surrounding the variant and 2) if there is differential binding between the alleles in individual subjects heterozygous for the risk variant at this locus.
- Reporter constructs: We use cells either transiently transfected luciferase or stably transformed GFP plasmids to assess the ability of a section of DNA to promote gene transcription, enhance gene transcription under the control of a minimal promoter, or repress gene transcription under the control of a maximal promoter. For each assay, reporter luminescence or florescence is compared to an internal control as well as a positive control. We compare the empty vector to the vector with DNA including the variant of interest in the context of the flanking approximately 1000 bases as specifically defined by relevant functional genomics data.
- Quality Control for experiments: Other controls will be encouraged as detailed throughout the descriptions of core experimental strategies supported. In cases in which an investigator is having difficulty using reagents designed for their specific experiment, we will provide reagents from projects that have been previously successful. In addition to suggested controls, the FGC will assist with the interpretation of experiments and will give example results (raw and processed) for investigators to use when assessing the specific results of their experiments.
- Genome-editing experiments and reagents: After establishing a strong rationale for plausible causality using other experimental strategies, the FGC will assist investigators in creating pairs of cell lines that are genetically identical with the exception of a single base change (the disease variant allele). We will achieve this through genome editing with or without homologous recombination (HR) using the CRISPR/Cas9 system.
Please contact Leah Kottyan for more information about project initiation.