Director: David A. Bernlohr, Ph.D.
Associate Director: Douglas Mashek, Ph.D.
In order to understand the mechanisms that underlie the etiology of obesity, it is critical to develop a comprehensive description of the basic biochemical and molecular events that ultimately lead to the deposition of long chain fatty acids. With the availability of such a description, one can develop alternative logical approaches to the treatment and prevention of obesity. The Obesity and Energy Metabolism Core provides Minnesota Obesity Center investigators with the latest tools for developing and evaluating gain-of-function and loss-of-function models and the techniques to assess the consequences at the level of gene and/or protein expression. The Core facilitates rapid and cost-effective acquisition of research results of participating investigators by providing access, training, and expertise in key molecular technology platforms used in obesity research. For this purpose, Core services are divided into four main components:
Gene Expression Analysis
The Gene Expression Analysis Facility provides MNOC investigators the opportunity to use adenovirus and lentivirus gene transfer vector technology in their research. Applications include transient or stable, long-term regulated expression as well as "knockdown" studies utilizing shRNA technology. The specifics of each service are provided below. All cloning will be based on Invitrogen's Gateway system.
Ideal for transient, high-level expression of protein of interest in dividing or non-dividingmammalian cells. Advantages include the generation of a high-titer virus following rounds of amplification and the ability to use the virus in vitro or in vivo following purification.
A flexible, pseudotyped viral platform with a broader host range compared to adenovirus facilitating stable, long-term high level tetracycline-regulated gene expression in dividing or non-dividing mammalian cells.
An artificially designed class of siRNA that offers potent and specific inhibition of eukaryotic gene expression. The VPF utilizes a viral based system developed to express the shRNA in mammalian cell constitutively (U6 promoted) or regulated by tetracycline (H1 promoted). Available in either adeno or lenti viral systems.
Specific responsibilities of Investigator and VPF
The investigator will download a Virus Production Request form (below) and meet with Rocio Foncea to discuss plans and timing of virus production. The VPF will provide reagents, vector backbone and instructions for cloning of gene of interest or double stranded oligonucleotide (for shRNA) into appropriate entry vector. The investigator is responsible for correct construction of entry vector and because this construction is crucial for downstream reactions and applications, a combination of sequencing, PCR and diagnostic restriction enzyme analysis is required.
The VPF will take a confirmed entry vector containing the insert of interest and perform recombination to generate appropriate adeno or lenti virus. It is the responsibility of the VPF to produce recombinant virus, to establish its titer and any agreed upon amplification, purification or concentration. The virus will then be delivered to the MNOC user. A stock of the virus will be kept within the facility. Contact between investigator and the VPF will continue as needed to the satisfaction of the investigator.
Download a Virus Production Request form.
Email or mail to:
Rocio Foncea, PhD firstname.lastname@example.org
Department of Biochemistry, Molecular Biology & Biophysics
420 Washington Ave SE
Minneapolis, MN 55455
Real Time PCR
For MNOC users, funds are available for the purchase of oligonucleotides to study expression of target genes using real-time PCR. An oligonucleotide library is established and available containing information about target gene, sequence, optimized PCR conditions as well as contacts in specific laboratories for additional information. Oligonucleotides purchases will be handled through the MNOC to ensure maintenance of the library.
To order oligonucleotide
The Facility will provide access to a Roche light-cycler as well as instruction on use and data evaluation. Reagents for these experiments are the responsibility of the investigator.
Real time primer list
Animal Physiology and Energy Metabolism
The Molecular and Cellular Basis of Obesity Core also assists investigators with measures of energy expenditure in small animals using indirect calorimetry. The equipment (Columbus Instruments) measures oxygen consumption and carbon dioxide production in awake, freely moving rats and mice, and the data can be used to determine energy expenditure. Physical activity levels can be measured concurrently using a beam break apparatus (Med Associates) fitted around the cage. Body composition measurement for rodents is also available. The equipment, EchoMRI (Echo Medical Systems) provides a non-invasive means and non-anesthesia means of providing fat and lean mass in rodents. Measurement time is 1-5 minutes per animal.
Adipocyte Cell Biology (ACB)
Dr. James Kirkland, MD, PhD, is Professor and Director, Robert and Arlene Kogod Center on Aging Research, Mayo Clinic. He is an internationally recognized expert on adipocyte cell biology and preadipocyte differentiation. He will be leading the Adipoctye Cell Biology subcore, providing differentiating human preadipocyte cells to MNOC users. This is a particularly important resource for the Core, allowing expansion of its base of cell lines beyond the standard 3T3-L1 model to include a range of human preadipocytes. Since several regulatory signaling systems differ between murine and human cells (e.g., natriuretic peptide signaling and lipolysis), the availability of human cells is a major technology advancement. This core offers two experimental cell culture systems; the well-defined 3T3-L1 murine adipocyte cell system and the human differentiating preadipocyte cell system. The 3T3-L1 system is well characterized, easy to use and the Core has from its history a repository of antibodies, primers and other reagents that can be used for characterization. The Kirkland lab brings to the MNOC the experience and core that he developed through the Boston Obesity/Nutrition Research Center. Although the use of the human preadipocyte cell lines is not currently as widespread, they offer unique opportunities that the 3T3-L1 system does not including gender, depot and BMI-based information from the human donors. This resource is unique and brings a new tool to the table for MNOC investigators. These cells are a national resource and represent a mechanism for the MNOC to connect to other NORCs.
Molecular Metabolism Journal Club
Weekly on Mondays at 3:30 PM
Room G205 Mayo--the NMR Conference Room