Obesity and Energy Metabolism Core
Director: David A. Bernlohr, Ph.D.
Co-Director: Howard C. Towle, 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:
Virus Production
The Virus Production 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.
Adenovirus
Ideal for transient, high-level expression of protein of interest in dividing or non-dividing mammalian 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.
Lentivirus
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.
shRNA
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 Candace Gename 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:
Candace A. Gename gena0006@umn.edu
Department of Biochemistry, Molecular Biology & Biophysics
7-178 MCB
420 Washington Ave SE
Minneapolis, MN 55455
Microarray Analysis
The Core provides support and expertise in the use of Affymetrix Gene Chips in conjunction with the Academic Health Center’s Biomedical Genomics Center. Center investigators receive access to chips and support at a reduced rate plus consultation on statistical data analysis from the Core.
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 oligos, please download and complete an ordering form and email to Candace A. Gename at gena0006@umn.edu.
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.
Small Animal Indirect Calorimetry
The Obesity and Energy Metabolism 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.
Real time primer list
Advancements in Obesity and Energy Metabolism |
