Gene Targeting & Knockout Core PDF *MNA User Authentication Required

Director
Demetri D. Spyropoulos, Ph.D.
Associate Professor of Pathology and Laboratory Medicine

Contact Information

Demetri D. Spyropoulos
Phone: 843-792-1625
E-Mail: spyropdd@musc.edu
Location: Children's Research Institute

Description
This Shared Resource consists of 4 facilities: Transgenic, Gene Targeting and Knockout, Xenograft, and Carcinogenesis. Transgenic and Gene Targeting facilities are essential genetic tools for studying mammalian gene functions in vivo, by testing the over-expression and loss-of-function mutations in animal development and in tumor susceptibility, respectively. In addition, Transgenic technology can be used in generating tissue-specific expression vehicles that can be utilized in creating conditional knockouts, either by Cre recombinase or by siRNA expression. Xenograft of human cancer cells or tissues into immune deficient mice is an indispensable step in correlating in vitro cell biology studies of cancer biology with tumorigenicity in vivo. The tumor grown in mice can also serve as a realistic test model for new therapeutics. For studying specific cancer types, the Carcinogenesis facility is able to generate several organ-specific cancers in rodents; they are invaluable tools for testing the function and therapeutic values of specific genes and therapies. The Cancer Animal Model Shared Resource combines the existing expertise of these four facilities to form a comprehensive service to assist cancer researchers at HCC.

The overall goal of Gene Targeting and Knockout component of the Animal Models Shared Resource is to provide the means by which researchers can learn and apply cutting edge technology to the molecular analysis of mammalian gene function. The specific aim of this facility is to create "knockout mice" through the utilization of DNA-, stem cell-, and embryo-manipulation procedures. Molecular cloning techniques are employed to clone and manipulate DNA sequences. Homologous DNA recombination in cultured embryonic stem (ES) cells is employed to generate "targeted" ES cells (i.e. ES cells carrying the replacement of specific chromosomal DNA sequences with cloned DNA sequences). Embryo manipulation techniques involving the targeted ES cells are employed to generate chimeric mice, which are then used to generate the knockout mice.

Services

• Mouse tail DNA Preparation
• ES Cell DNA Preparation
• Mouse Genotyping
• ES Cell Electroporation
• Blastocyst Injection
• Embryo Cryopreservation
• Blastocyst Transfer
• Chimera Maintenance and Breeding
• Mouse Weaning and Tailing
• Timed Matings
• Various Mouse Surgical Procedures
• Knockout Offspring Maintenance
• Mouse Purchase
• Breeding Mice

Equipment

• 6 ft Baker SterilGARD Class II Hoods
• 2 Forma CO2 incubators
• Nikon dissecting scope equipped with a Hamamatsu videocamera on a Vibraplane isolation table
• Zeiss Axiovert 10 injection scope with cooling stage and Narishige Micromanipulators on a TMC isolation table
• RM6 Lauda cooling water bath
• Leitz Labovert ES cell scope equipped with a Leica photomicrographic system
• Biorad Gene Pulser
• Taylor-Wharton cryostorage unit
• Planar Controlled Rate freezer for embryo cryopreservation
• Holton LaminAir dual workstation hood
• Water baths
• Refrigerator/freezer