Foundation for Advanced Education in the Sciences

Co-Sponsored by the NIH Center for Regenative Medicine (CRM)

Gene engineering provides the ability to manipulate gene expression in a desired cell type. In order to realize the full potential of stem cells, the development of tools to modify targeted genes is paramount.

Transcription Activator-Effector Like Nucleases (TALENs) are artificial restriction enzymes that allow for efficient, programmable, and specific DNA cleavage. This recent technology offers precise engineering of your gene of interest and has been shown recently to specifically mediate site-specific genome modification in mammalian cells including iPSCs.

During this 3-day workshop, participants will learn the principles and methods of TALENs and given the base knowledge to create their own TALENs.

Topics: Lecture: Introduction of Designer Nucleases, From ZFN to TALEN (A Review of Moving From ZFN to TALEN, Various TALEN Platforms and their Applications Available Today); Gene Deletion in Mouse B cells and ES cells Using TALENs (Protocols of Efficient Screening of Targeted Cells, Discussion of Gene Targeting Strategies); DNA Repair Mechanisms (Scientific Background Behind the Technology, Case-Study Discussions Along With Time to Discuss Circumventing Technical Issues and Troubleshooting); TALEN-Mediated Knock-in Reporter in Human iPS Cells; Using TALEN in Human, Mouse and Zebrafish Research

Laboratory: Introduction of TALEN Design Website and Software; Set up Golden Gate TALEN ligation; Ligation Results, Pick Colony; Computer Design of Your Own TALEN; Miniprep, Digestion; Set up Final Golden Gate TALEN Ligation

Class Limit of 24

Benefits for Early Registering:

Register by May 1, 2013 to take advantage of the early registration discount ($895). The registration fee will increase to $995 on May 2, 2013

First 15 Registered Participants will receive at no additional charge from the NIH Center of Regenerative Medicine (CRM) a Safe Harbor TALEN ($200 commercial design), a donor vector ($1,000, commercial design), and a cell line ($200 - $500, commercial order) Note: MTA must be signed in advance.

Should you wish to use the course to start your own project, participants can submit a target site and we will help you design it during the course.

One Submitted TALENs will be used to practice hands-on TALEN Assembly Experiment.

Co-Sponsored by the NIH Center for Regenerative Medicine (CRM)

Recent advances in generating iPSCs now allow for their derivation from blood. This recent advance enables basic and clinical researchers to reprogram a blood cell into an iPSC and then further differentiate into any cell type. This capability allows researchers to develop "disease in a dish" paradigms to investigate disease and therapy mechanisms.

In this one week workshop, participants will learn how to generate iPSC from blood samples using a non-integrating approach. Due to the length of this procedure (iPSC generation ~3-4 weeks etc.) starting material (CD34+ cells or mononuclear cells) will be provided for each investigator and only critical stages will actually be performed during the laboratory portion of the workshop.

In addition to learning how to culture cells and reprogram blood cells into iPSCs, we will also present some of the latest methodologies for directing differentiation of these iPSCs into different lineages (e.g. hepatocytes or cardiomyocytes). Therefore, this course will package together the essential methodology to take a CD34+ cell isolated from blood, reprogram this cell, and then direct differentiation into multiple different lineages.

Topics: Introduction to Mouse and Human Induced Pluripotent Stem Cells; Cellular Reprogramming Methodologies, Reprogramming from Blood; Techniques Used for Blood Cell Purification and Culture; CD34+/WBC Purification and Culture; Transfection of Human CD34+ Cells With Lenti/Sendai Viruses; Human Pluripotent Stem Cell: Growth Conditions, Handling Methods, Expansion and Cryopreservation; Differentiation of IPS to Major Lineages; Preparation of Cells From Embryoid Bodies To Study Differentiation; Differentiating IPSC to Blood Cells, Endothelial Cells, Neural Cells, Hepatocyte Lineage; Stem Cell Marker Analysis

Benefits for Early Registering:

First 15 registered participants will receive an iPSC line generated by NIH CRM, a $20,000 value (must register before May 17, 2013)

Register by June 28, 2013 to take advantage of the early registration discount ($995). The registration fee will increase to $1,095 on June 29, 2013

Class size will be limited to 24 (waiting list anticipated)

This lecture and laboratory course is designed to provide the novice with an introduction to recombinant DNA technology. An approach emphasizing both principles and methodology provides the scientist with the essential fundamentals needed for gene cloning and for an appreciation of the strategies of recombinant DNA technology.

Topics: The Power of Recombinant DNA Technology-its advantages and applications; Recombinant DNA Safety Guidelines; Vector-Host Systems; Propagation and Maintenance of Bacterial Strains and Viruses; Isolation of Cloning Vector-bacteriophage and Plasmids; Construction of Genomic Libraries; Construction of cDNA Libraries; Extraction and Purification of mRNA from Eukaryotic Cells; Synthesis of cDNA; Screening for Differentially Expressed Genes; Microarrays; Differential Display; Nucleic Acid Sequencing and Site Directed Mutagenesis; Restriction Enzymes; Analysis of Nucleic Acids by Gel Electrophoresis; Insertion of the cDNA with Vectors; Transfection of Bacteria with Recombinant DNA; Identification and Analysis of Recombinant DNA Clones; Vector Systems which allow for the Expression of the Cloned Fragments in the Bacterial Host; Special Expression Vector Systems: Strategies in Applying Recombinant DNA; Gene Therapy

From its conception in 1983 to its modern day use in a myriad of clinical and research applications, the Polymerase Chain Reaction (PCR) has revolutionized modern molecular biology. This lecture and laboratory course will focus on the Polymerase Chain Reaction and its applications in basic molecular biology research, genetics, molecular pathology, including cancer and genetic diseases and identification of viral, bacterial and other pathogens.

Topics: Topics include Basic PCR, Dissection of the PCR Cycle, Primer Design and Usage including Tools for Primer Design, Thermostable DNA Polymerases, Optimization, PCR Additives, Contamination Control, Reverse Transcription PCR (RT-PCR) PCR Mutagenesis, Cycle Sequencing, 5' and 3' RACE PCR, Multiplex PCR, Clinical and Forensic Applications, Practical Modifications to PCR.

Laser Microdissection systems allow for the procurement of specific populations of cells from tissue and cytology and live cell culture samples containing heterogeneous populations of cells. The specificity of analyses is therefore much more representative of the disease process being studied. This approach to microdissection ensures that biological molecules, such as RNA and DNA and proteins, remain undamaged during the microdissection process. Downstream molecular analysis of these molecules produces accurate and assured results that have led to over 2,000 peer-reviewed publications by independent researchers.

In this five-day training program, participants learn to prepare tissue specimens for microdissection, then select and acquire homogenous cell populations using the mmi-CellCut, Leica LMD, Arcturus XT, and PALM microdissection systems. Instruction emphasizes operation of these LM systems, appropriate tissue handling and sample preparation for subsequent DNA, RNA or protein analysis, and methods for proper molecular extraction. Lecture and detailed instructions to prepare samples for several downstream molecular analyses are presented.

Topics: Overview of Laser Microdissection Technology (History, Theory, Applications); Tissue Sample and Slide preparation; Project set up and QC of microdissected samples, genomic analysis, mRNA analysis including quantitative RT-PCR gene expression analysis and microarrays from microdissected tissue samples, microRNA analysis from microdissected samples, and proteomic analysis from frozen and formalin-fixed, paraffin-embedded microdissected samples using Mass Spec. Special microdissection techniques such as immuno-guided LM and live cells dissections will be also covered. The lectures also include examples of actual scientific and clinical applications of LM-based studies. The hands on sessions include tissue slide preparation and histology review for LM, and practice on Arcturus XT, PALM, Leica and mmi-CellCut platforms.

The emerging field of Clinical Proteomics refers to the application of proteomic technologies to investigate protein expression differences in clinically obtained biological samples. A consequence of these approaches has been the renewed interest in identifying new biomarkers which may useful for the diagnosis of disease or monitoring of patient response to therapy. Researchers involved in Clinical Proteomics face numerous methodological, analytical, and statistical challenges that need to be addressed in order for successful completion of projects. Additionally, large scale biomarker discovery efforts include the coordination of many different disciplines, such as biochemistry, proteomics and mass spectrometry, clinical chemistry and bioinformatics. In this course, students will be exposed to many of the challenging aspects of biomarker discovery projects, as well as to the numerous analytical platforms that may be employed.

Topics: Study Design and Sample Collection/Storage of Clinical Samples; Sample Handling and Preparation (Chromatographic methods to reduce the complexity of the sample, such as ion-exchange chromatography, immunoaffinity depletion); Quality Control and Reproducibility in Measurements; Analytical Tools for the measurement of quantitative difference between clinical samples: 2D gel electrophoresis, including DIGE; MALDI/SELDI-TOF-MS for Serum Protein Profiling, Multiplex Protein/Cytokine Arrays, Differential isotopic labeling, such as ICAT, SILAC Quantitative Data Analysis of 2D and Mass Spec Data Sets; Multivariate Statistical Models; MS Protein Patters vs. Protein Panels for Diagnostic Determination

Conventional PCR has revolutionized the detection and analysis of nucleic acids. However, one of its major limitations has been the inability to accurately quantitate the amount of product, which reflects the amount of starting material, due to differing plateau effects among multiple samples. The need to accurately determine quantitative changes in gene expression has led to the adoption of real-time RT-PCR as the method of choice not only for quantitative gene expression but also for validating results obtained from array analyses and other techniques that evaluate gene expression changes on a global scale. This lecture/laboratory course is intended for those who have a fundamental background in PCR and will address the basic chemistries of real time PCR and the many platforms available.

Lecture and Laboratory Topics Include: Introduction to and Brief History of Real-Time PCR; Sample Processing: DNA/RNA Extraction and Purification for Real-Time PCR; Probe Chemistries and Real-Time Instrumentation including Amplification using Three Different Platforms; Assay Development; Quantitative Real-Time PCR; Multiplex Real-Time PCR; Array qPCR; Droplet Digital PCR (ddPCR).

This course will introduce students to bioinformatic analysis of next generation sequencing data, particularly for DNA-seq, RNA-seq, CHIP-seq, and epigenomics. The course will be comprised of lectures and hand-on sessions. Lectures will cover background knowledge and survey various software programs. For hand-on sessions, command line tools will be presented and the galaxy web based platform will be used to analyze primary data. Cloud computing, genomic databases, and de novo assembly will be surveyed.

Topics: Overview on Bioinformatics Tools: Galaxy (Pre-processing, Format Conversion, etc.), Databases and Tools, SNP Callers, Cloud; RNA-seq; CHIP-seq; Epigenomics; Cloud Computing; 10k Genomes; Data Visualization; Comparative Genomics and Genome Alignment (biology + software); Tutorial & Laboratory for de novo Assembly; RNA-seq, DNA-sequencing.

This course will introduce students to bioinformatic analysis of next generation sequencing data, particularly for DNA-seq, RNA-seq, CHIP-seq, and epigenomics. The course will be comprised of lectures and hand-on sessions. Lectures will cover background knowledge and survey various software programs. For hand-on sessions, command line tools will be presented and the galaxy web based platform will be used to analyze primary data. Cloud computing, genomic databases, and de novo assembly will be surveyed.

Topics: Overview on Bioinformatics Tools: Galaxy (Pre-processing, Format Conversion, etc.), Databases and Tools, SNP Callers, Cloud; RNA-seq; CHIP-seq; Epigenomics; Cloud Computing; 10k Genomes; Data Visualization; Comparative Genomics and Genome Alignment (biology + software); Tutorial & Laboratory for de novo Assembly; RNA-seq, DNA-sequencing

THIS COURSE IS CLOSED FOR REGISTRATION. Please email the FAES Assistant Registrar at candice.allar@nih.gov if you would like to be added to the waitlist.

The NINR Fatigue/Sleep Methodologies Boot Camp, part of the NINR Symptom Research Methodologies Series, is a one-week intensive research training course at the National Institutes of Health (NIH) in Bethesda, Maryland. Sponsored by the National Institute of Nursing Research (NINR), the course is administered by the Foundation for Advanced Education in the Sciences (FAES) as one of the Bio-Trac programs.

The Fatigue/Sleep Boot Camp will provide a foundation in methodologies for use in research. The purpose of the course is to increase the research capability of graduate students and faculty. The course will feature lectures by distinguished guest speakers, classroom discussion, and laboratory training.

The course is provided by the NINR at no cost. Attendees are required to pay for housing, food, and transportation expenses incurred during program participation.

Course Topics

This course will cover a number of topics relevant to fatigue and sleep research including fatigue/sleep measurement, treatments, and genetics. For additional details, please see the agenda.

Course Objectives
•Increase knowledge in fatigue/sleep methodologies for use in research
•Discuss strategies for incorporating novel methods into research proposals

The Summer Genetics Institute (SGI) is a tuition-free one-month intensive research training program at the National Institutes of Health (NHI) in Bethesda, Maryland. Sponsored by the National Institute of Nursing Research (NINR), the SGI provides participants with a foundation in molecular genetics appropriate for use in research and clinical practice. The program seeks to increase the research capability among graduate students and faculty and to develop and expand clinical practice in genetics among clinicians. Administered by the Foundation for Advanced Education in the Sciences (FAES) as one of its Specialty Bio-Trac programs, the SGI features lectures and hands-on laboratory training. On completion of the program, participants receive eight hours of graduate-level college credit from FAES.

Prior approval from NINR is required to register for this program.

The objective of this lecture and laboratory course is to provide investigators with information on chemical approaches to the isolation, purification and characterization of antibodies and antigens. Special emphasis will be given to monoclonal antibody formation, assay, and characterization.

Topics: The Immune Response; Immunoglobulin Structure and Diversity; Immunogenetics; Antigenic Determinants; Idiotypic Network; Application of Monoclonal Antibodies; Cell Fusion; Myeloma Cell Lines; Screening for Monoclonal Antibodies (ELISA, Immunofluorescence, RIA); Cloning; Purification and Characterization Approaches; Human Monoclonal Antibody Formation and use; Crystal Structure; Purification of Antibodies from Serum, Ascites, and Culture Supernatant; Analysis of Antibody-Antigen Reactions by Immunoprecipitation and Equilibrium Dialysis; Affinity Determinations; Production of Idiotypic Antibodies, Antibody Fragments, and Novel Antibodies; Peptide Carriers and Antigen Synthesis; Radioreceptor and Crosslinking Assays for Cell Surface Receptors; Immunocytochemistry; Recombinant DNA Approaches.