Molecular Biology Core
Equipment & Facilities
Transgenic Mouse Program
The University of Michigan Transgenic Animal Model Program has a large collection of state-of-the-art equipment for mouse embryo collection and microinjection, and tissue culture facilities for ES cell growth and manipulation. Program laboratories include an embryo microinjection laboratory of 993 sq. ft. in Medical Science Research Building I, and ES cell culture laboratories of 194 sq. ft. and 566 sq. ft. in Medical Science Research Building II. These laboratories are in close proximity to each other in the University of Michigan Biomedical Core Laboratory space adjacent to the animal housing area where the Transgenic Program has a dedicated specific pathogen free mouse room with a capacity up to 500 mouse cages. Dr. Saunders’ office is down the hall from the Program laboratories, and the director offices are in connecting buildings: Dr. Camper in Medical Science Research Building III and Dr. Samuelson in Medical Science II. The specialized equipment in the Transgenic Program is listed below.
Mouse Embryo Lab Equipment: Five microinjection work stations with Nikon microscope and Narishige micromanipulators are set up for DNA injection into fertilized eggs and ES cell injection into blastocysts. One work station includes a video monitor and camera to view micromanipulations. Three mouse embryo retrieval and surgery workstations are in the same room, which include laminar flow benches, microscopes, and surgical equipment. Support equipment for the microinjections includes pipet pullers, microfuges, incubators, and a Prime Tech PMM1 150 FU piezo drill. A programmable embryo cryopreservation cooler is available to freeze embryos for storage in liquid nitrogen. An AutoGen 740 Robotic workstation is used for purification of genomic DNA from mouse and rat tail biopsies.
Tissue Culture Equipment: Cell culture workstations are available for up to 7 people to work with ES cells. Equipment includes biological safety cabinets, incubators, centrifuges, Bio-Rad Gene Pulser electroporator, microscopes for cell culture and chromosome analysis, Milli-Q PF water purification system, autoclaves, -80oC freezers, and liquid nitrogen storage containers. A Forma 1535 alarm/monitor of the type used in blood banks is used to continuously monitor the incubators and freezers.
Viral Vector Program
The Vector Program occupies 1,475 square feet of wet laboratory space on the third floor of Medical Sciences Research Building II. This space is centrally located near laboratory space occupied by Peptide Center members residing in Medical Science Buildings I and II and Medical Science Research Buildings I, II and III. The Vector Program space is divided into three physically separate wet laboratory modules. One 750 square foot module is configured specifically for the construction, isolation and purification of recombinant adenovirus. This module contains 3 laminar flow tissue culture hoods, four dual chamber CO2 incubators, ultracentrifuge, BioRad iCycler and allows 4 researchers to work simultaneously. Two hoods and incubator stacks have been reserved for core staff use. A second 225 square foot module in 3581 MSRB II is configured for the construction, isolation and purification of recombinant retrovirus. This module is equipped with one laminar flow hood and two CO2 incubator stacks, and an ultracentrifuge that is dedicated to retroviral studies. A third 500 square foot module in 3556 MSRB II has been configured to allow for the construction, isolation and purification of plasmid vectors and related molecular biological analyses. In addition, an adjacent common equipment area has been equipped with an environmental shaker for growth of bacterial cultures, as well as two -80ºC freezers and liquid nitrogen freezer for storage of viral stocks and cell banks. Together these modules provide the Vector Program with the space necessary to comply with biosafety requirements (BL2) for the use and production of these vectors as established by the University Michigan office of Occupational Safety & Environmental Health and the NIH. It establishes a Program facility that can provide investigators with a variety of vector systems for use as in vitro and in vivo reagents.
Microarray Gene Chip Program
The Microarray Gene Chip Program is a component of the Michigan Microarray Core and occupies a 500 sq. ft. laboratory space in Room 7410 of the Cancer Center and Geriatric Center (CCGC) of the University of the Michigan medical school campus. Available equipment includes the large-scale liquid handling Packard MultiPROBE IIex robot, the Axon GenePix 4000A microarray scanner, a Fluidics Station 450, three Hybridization Oven 640s, and a GeneChip Scanner 3000, an Agilent 2100 BioAnalyzer, an ABI 7900HT quantitative PCR and SNP genotyping apparatus, a large -80oC freezer for the storage of cDNA clones, and seven separate computer workstations dedicated to different pieces of equipment or data analysis. Other equipment includes four MJ thermalcyclers, several horizontal gel electrophoresis units and power supplies, microcentrifuges, incubators and bacterial shakers, refrigerators and freezers, a DNA speedvac, vortexes, balances, hotplates, pH meter and smaller lab equipment (micropipettors,etc.). A biostatistical area comprising the office area for Mr. James MacDonald and three computers dedicated to statistical analysis of microarray data (including a Linux box) are also housed in the facility.
Molecular Techniques Training Program
Advisory session will be carried out by e-mail for short questions or approval of protocols or by appointment in her office for more detailed questions. Practical training sessions will be carried out in her lab (1700 sq. ft). Dr. Merchant’s laboratory is in the Medical Science Research Building I on the 3rd floor. It is equipped with a Wallac Victor 3 Multilabel luminometer that can read 96 well microtiter plates, a Wallac Trilux Liquid Scintillation and Luminescence Counter, a BioRad Rotofor purification apparatus, a Quantitative PAGE apparatus, a liquid Nitrogen storage container for cell lines, two –80°C freezers and access to a dark room.
Advice on ChiP Assays
Dr. Merchant recently completed a sabbatical in which she performed chip assays using antibodies to ZBP-89 and -99. Some of this work was published in a collaborative study with Dr. Gardner of the NCI with whom she completed her sabbatical (Smith et al., PNAS 2004). Together with Dr. Bai, she has continued studying the regulation of the p21waf1 promoter by ZBP-89 using ChiP. An example of this ChiP assay is shown in Fig 1. Both the ChIP Kit from Upstate Biotechnology and ChIP protocol developed by Dr. Gardner has been used with great success. She has found that the quality of the antibodies and the efficiency of the DNA extraction to be critical elements in the success of the assay. The method developed by Dr. Gardner is detailed below under the section H on Specialized Methods. We plan to offer both technical advice and practical training to those who want it. However polling the center investigators revealed a 6 to 1 preference for advice. Practical training will be carried out under the direct aegis of Dr. Bai. Figure 1 below is an example of the quality of a ChIP assay performed with the Upstate Kit.
Fig. 1. HCT116 cells were treated with 2.5 mM sodium butyrate for 1h and soluble chromatin was prepared. The immunoprecipitation was performed with anti-ZBP-89 IgG and rabbit IgG control. The final DNA extractions were amplified using pairs of primers that cover the regions flanking p21waf1 transcription start site as indicated. The input is 0.2% of the total reaction mixture. The results show ZBP-89 inducible binding to the region –322 to –33 and identified a novel constitutive binding site at +251 to +500.
Availability of Luminometer
We are requesting funds for a service contract on the luminometer used currently by six Center investigators. The luminometer is the Wallac Victor3 1420 Multilabel luminometer (by Perkin Elmer) and is capable of dual substrate-injection. Due to heavy use by the six investigators, we need to maintain the equipment in good working order. Even within the first year the machine has required servicing twice.