Mentoring Vision Research in Oklahoma
COBRE
Department of Ophthalmology
University of Oklahoma Health Sciences Center
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Proteomics/Bioinformatics

Image Acquisition and Production

Animal Core

Microinjection

Microarray

Molecular Biology
MOLECULAR BIOLOGY CORE FACILITY

The Molecular Biology Core Facility supports investigators at OUHSC in their research programs that utilize transgenic animal models. This core facility is composed of two basic components: 1) an Embryo Freezing and Storage Facility and 2) a Transgenic Frog Facility. Dr. Allan F. Wiechmann is the Director of this Molecular Biology Core. His research laboratory is directly adjacent to the Transgenic Frog Facility, and is in very close proximity (100 feet) from the Embryo Freezing and Storage Facility. Two technicians with expertise in appropriate areas perform the daily tasks to operate the facility, under the direct supervision and participation of the Director.

Embryo Freezing and Storage Facility

The Embryo Freezing and Storage Facility is located on the 5th floor of BMSB, and is within 100 feet of the Director's laboratory. This facility enables us to preserve transgenic lines for long term by freezing, which reduces the cost for maintaining the transgenic lines for the investigators and insures against major disasters, such as flooding, which occurred recently in transgenic housing facilities in Houston.

Embryo freezing, although not difficult, requires proper conditions. Our facility enables slow, gradual freezing using a protocol that has been optimized for embryos as well as equipment to for long-term maintenance of frozen embryos. Whenever the need arises, embryos are removed from the freezer and reimplanted in foster mothers to re-establish the line. Embryos can also be shipped to collaborators throughout the world without major difficulty and can be re-implanted on site. Also, since we are establishing a re-implantation facility, embryos can be delivered to OUHSC and re-implanted on site to avoid the introduction of murine diseases that may be indigenous to colonies outside OUHSC. The day-to-day operation of this facility is supported by a 30% effort by an experienced technician, and is overseen by several members of the vision core, including the Director.

Transgenic Frog Facility

The African clawed frog, Xenopus laevis has served as a major model for the study of embryonic development because of features such as external development, large size, identifiable blastomeres, and its ease of culture in vitro. These features have enabled Xenopus to produce a detailed cellular and molecular understanding of early patterning events, including an understanding of the role of specific extracellular growth factors, cell surface receptors, and intracellular signaling pathway components. Thus, Xenopus is ideally suited to provide critical breakthroughs in early embryonic patterning and cell fate determination, later development and organogenesis, oncogenesis, and cell biological and biochemical processes.

Recent advancements in transgenic Xenopus technology make this an ideal model for the study of expression of genes and their function. Transgenic frogs can be generated much faster and cheaper than transgenic mice. In this method, foreign DNA is introduced directly into the sperm genome before fertilization by a method called restriction enzyme-mediated integration (REMI). The transgenic sperm nuclei are then injected into oocytes, producing transgenic embryos that are non-mosaic and therefore do not have to be bred before analysis. Because many tissues and organs, especially the retina, develop quickly in Xenopus, analysis of the genetic manipulation can be done very quickly. Furthermore, the transgenic embryos carrying promoter-reporter constructs show spatially and temporally appropriate reporter-gene expression patterns. Green Fluorescent Protein (GFP) is most often used as the expression marker.

Major Equipment

1. Embryo Freezing and Storage Facility

The Embryo Freezing and Storage Facility enables users to store mouse embryos for later implantation. This eliminates the need to house animals simply for the purpose of maintaining a genotypic line. It also insures against sudden disease in the mouse colony, as well as natural disasters.

a. MVE XLC 810 HE Freezer. This is a LN2 storage container with a guaranteed vapor temperature of -150oC. It automatically fills and is continuously monitored by a TEC 2000 electronic controller. This is used to store frozen embryos for extended periods of time.

b. Auto Liquid Nitrogen Tank Switcher. This is attached to the MVE freezer, and detects whether or not the liquid nitrogen source tank is empty. When this occurs, the unit selects the other tank for uninterrupted filling of the freezer.

c. TS Scientific Kryo 360-3.3 Biological Freezer. This system is designed to start samples at ambient temperature, and then uses a selected freezing protocol to prepare samples for long-term storage. It is interfaced with a computer, and uses Delta T software to control freezing and thawing cycles.

d. R1 LN2 delivery system. This system uses a pump and power supply to enable it to serve as a liquid nitrogen source for the Kryo 360.

e. IQ800 Power Supply. This is an uninterruptible power supply for the Kryo 360.


2. Transgenic Frog Facility

The transgenic core is housed in two separate rooms on the 5th floor of BMSB. The frog housing area occupies 130 sq. ft adjacent to the Director's laboratory, and the major procedures (e.g.; REMI reactions, microinjections, micropipette formation, and embryo staging area) are performed in a dedicated 120 sq.ft. area in the Director's research laboratory. All of the components are recently purchased, state-of the art equipment.

There are two Oocyte Injection Stations in the core. This enables two workers (the technician and director) to inject sperm nuclei into oocytes simultaneously. This is required since the time period in which the sperm nuclei preparations are viable will exceed the amount of time required for only one worker to inject the 2,000-4,000 oocytes in each experiment. Each injection station is comprised of the following components:

a. Harvard Apparatus Pump 11 Single Syringe Pumps. These provide controlled delivery of the sperm nuclei into the injection needles that are used to inject frog oocytes. The pumps are set at a specific flow rate, and the controls the precise and reproducible regulation of injection pressure and time.

b. Harvard Apparatus Manual Control Micromanipulators. This apparatus provides the stability needed to perform multiple nuclear transplantations. The frog oocytes are very small, and the injections must be performed while viewing under a dissecting microscope. The Micromanipulators provide the precision needed for injecting the oocytes.

c. Two VWR Fiber-Lite Illumination Systems. These fiber optic light units provide cool light to the viewing area under the dissecting microscopes, to permit precise injections of oocytes, and staging of transgenic embryos.

d. Two Zeiss Stemi 2000 Stereo Microscopes. The dissecting microscopes are used to view the oocyte injections and for staging of the transgenic tadpoles. It should be emphasized here that these dissecting microscopes do not fulfill the requirements for fluorescent screening of transgenic tadpoles. The inverted fluorescent microscope requested in the proposal is used for an entirely different purpose, and have many specialized features (e.g.; fluorescence, inverted configuration for viewing animals in wells, range of magnification, etc.) that is lacking in the dissecting microscopes at the injection stations.

The other major components of the Transgenic Frog Facility are:

e. Narishige Model PP-830 Microelectrode Puller. The pipette puller enables us to prepare micropipettes that have the precise angles and size required for optimal injections of oocytes.

f. Research Instruments Limited Model MB3-T Turbo Microbeveller and power supply. The microbeveller is used to create the precise angle and bore size of the micropipettes. This provides us with very high quality micropipettes that provide higher efficiency of injections, and a higher number of transgenic animals.

g. VWR Model 1300U High Temperature Oven. This oven is used in concert with a glass desiccator to siliconize the micropipettes, which is necessary to protect the sperm nuclei from disruption during the injection process. The oven is used to bake the micropipettes after they have been siliconized.

h. Two WWR Model 2005 Low-Temperature Incubators. One incubator is maintained at 18°C, and the other is at 22°C, which allows for the early embryo stages to be housed at the optimal temperature of 18°C, and then to promote the later stage animals to their higher optimal temperature of 22°C.

i. Precision Scientific 818 Low Temperature/Light Cycle Incubator. This environmental incubator is used to house froglets and adult frogs under precisely controlled cyclic lighting and temperature conditions. This protects the animals from inappropriate exposure to light or darkness.

j. Aquatic Habitats Z-Plex Stand-Alone System. This is an aquatic holding system that can house 100 adult Xenopus, or 1000-50,000 tadpoles, depending on their age. The system can contain 100 gallons of water within a footprint of 13 square feet. It is equipped with automated life support and provides a stable re-circulated environment for the frogs and tadpoles.

k. Four Aquatic Habitats Uniplex 28-Gallon Housing Tanks. These holding tanks are used to house the adult male and female breeder frogs, and non-transgenic froglets.