Molecular Biology/Molecular Genetics Core

Introduction

Virtually all biomedical research increasingly depends on, or can benefit substantially from, access to the set of experimental, and analytic tools often referred to as genomics. This term denotes a set of rapidly changing techniques for acquiring and interpreting large amounts of data relating to gene sequence, variation, expression and function. These approaches require special, costly equipment, skilled technical personnel and, frequently, intense computational treatment, all of which are inefficient or impossible to replicate in multiple individual laboratories. Many of these techniques relate to the high throughput assessment of genetic variation to evaluate possible genetic contributions to metabolic phenotypes of obesity. In addition, sophisticated tools for the manipulation of genes - by timing and organ-of-expression - in mice makes it possible to generate and test mechanistic hypotheses regarding energy homeostasis in definitive ways. It is clear that such techniques are necessary for modern biological approaches to complex phenotypes related to obesity. By making these sophisticated genomic techniques readily available to a large number of investigators without substantial individual investment of capital or technical expertise, investigators will be able to effectively utilize genomic tools of large scale gene expression or protein analysis, sequencing, genotyping and transgenic manipulation to study phenotypes in which they have expertise. These tools are particularly relevant to the genetic analysis of complex traits, of which obesity is paradigmatic.

Purpose of the Molecular Biology/Molecular Genetics Core

The overarching mission of the MB/MG Core is assist investigators seeking to discover the molecular bases underlying obesity and its comorbidities. To achieve this mission, the Core performs studies of material from humans and rodents to identify mutations in candidate genes, as well as rodent studies to define the functions of candidate genes by genetic manipulations using transgenic methodologies. Through collaborative efforts with the other Cores of the NYORC, the effects of defined genetic alterations in humans and rodents are thoroughly characterized for their effects on ingestive behavior, energy balance, body composition, and endocrine function. The role of candidate molecules in relevant tissues, such as neurons, adipocytes and pancreatic islets that are related to energy homeostasis can be sharply delineated by thorough and definitive experimentation on both human subjects and rodent models. Due to the establishment of close ties among the various Cores of the NYORC, opportunities to explore new genetic models of obesity are greeted with enthusiasm by the collaborators of the MB/MG Core.

The broad expertise of the MB/MG staff allows the MB/MG Core to make available to biomedical investigators a wide range of methodologies and reagents relevant to understanding the molecular physiology of obesity and energy metabolism in animals and humans. This core applies molecular genetic and molecular biological techniques to the mapping, cloning and functional characterization of genes related to obesity and its comorbidities (diabetes and atherosclerosis). The services of this Core are available to investigators new to obesity research, as well as to investigators working on obesity-related projects that can be enriched and extended by the use of the expertise and facilities of this core.

The objectives of this Core are to:

1. Perform molecular biological and molecular genetic experiments with and for qualified investigators.
2. Perform functional genomics analysis, such as brain, adipose tissue, muscle and pancreatic islet gene expression studies, related to obesity.
3. Instruct investigators on public access software (Internet Websites) that form a comprehensive suite of programs for molecular biology and molecular genetics (sites maintained by NCBI (ENTREZ, BLAST, OMIM, PUBMED), Whitehead Institute for Genome Research, Jackson Laboratories (MGD), Swiss Institute of Bioinformatics (EXPASY), Baylor College of Medicine (BCM Launcher), Sequence Navigator, Sequencher, Phrap and Paracel assembly software, TRANSFAC/MATInspector, and other software available through the bioinformatics facilities at Columbia (http://amdec-bioinfo.cu.genome.org).
4. Train investigators and technical staff in molecular biological and molecular genetic techniques.
5. Advise investigators in design and performance of molecular genetic studies in rodents and humans.
6. Maintain and distribute breeding colonies of animals with mutations and allelic variants at quantitative trait loci related to obesity and diabetes. This service includes advice in the creation of transgenic mice.

The allocation of services and reagents (described below) is made on the basis of the following criteria: access of these facilities will support existing NIH funded research; the services are likely to lead to the acquisition of data needed to apply for independent research funding; the services will advance knowledge in the field of obesity research and/or the services provide an educational venue for students. In many instances, there is a close collaboration between the investigator requesting assistance and the personnel of this Core.

Overview of Techniques and Services Offered by Molecular Biology/Molecular Genetics Core

1. Molecular Biological Techniques
   • Isolation and quantification of DNA
   • Isolation and quantification of RNA
   • DNA sequencing
   • Quantification of mRNAs by Northern blotting, semi-quantitative RT-PCR, and in situ hybridization
   • Generation of cDNA and genomic clones for obesity genes
   • Determination of gene structure - definition of intron-exon boundaries
   • Parallel Gene expression analysis with cDNA and oligonucleotide arrays
   • Use of siRNA
2. Molecular Genetic Techniques
   • Genetic mapping by linkage analysis
   • Physical mapping by computation and use of large insert libraries including C57BL/6J BAC library on hand.
   • Integration of genetic and physical maps
   • Quantitative trait locus mapping in rodents
   • Mutation scanning by denaturing HPLC
   • Development of high-throughput PCR-SNP assays for large-scale association studies
   • Genotype analysis with simple sequence repeat (SSR) markers and single nucleotide variants (SNP)
   • Genotype analysis in pooled or single samples by pyrosequencing
   • High throughput SNP analysis by FPTDI
   • Association studies of candidate obesity genes in human populations
   • Computational analysis of mouse and human genomes.
   • Representational oligonucleotide microarray analysis (ROMA)
3. Animal Models
   • Congenic mouse strains with leptin deficiency
   • Congenic mouse and rat strains with leptin receptor deficiency
   • Mice with Mc4r and Mgrn1 (mahoganoid) mutations
   • Transgenic and knockout mice
     • Consultation on transgene design
     • Library screening to obtain genomic clones from 129, DBA, and C57BL/6J strains
     • Provision of vectors for transgene and knockout constructs
     • Access to full service transgenic mouse facilities at both Columbia and AECOM
   • Training for performance of glucose and insulin tolerance tests in rodents

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