CORE SERVICES

CORE 1

GENETICALLY-DEFINED MICROBE CORE

John C. Kappes, Ph.D.

Charles O. Elson, M.D.

 Access

       Requests from Center investigators will be accepted on a first-come, first-served basis, subject to the requirement that the research be appropriate for the Center project and likely to contribute to its success.

Retroviral Unit (Dr. Kappes)   

1)      Molecular construction of gene expression vectors (preparation of DNA; restriction  analysis of DNA; insertion of gene; confirmation of gene expression; large-scale [Maxi prep] production of DNA.

2)  Validation of vector trans-gene expression (DNA transfection and confirmation of gene expression.                                   

3)     Packaging of the vector and cryopreservation of the virus vector stock (preparation of DNAs for packaging and Env constructs; transfection of mammalian cells; collection and preparation of culture supernatant [virus vector]; cryostorage).

4)     Concentration of the virus vector stock (large-scale transfection of mammalian cells; collection and preparation of culture supernatant [virus vector]; cryostorage).

5)      Generation of GFP-virions (preparation of DNA; large-scale transfection of mammalian cells; collection and preparation of culture supernatant [virus vector]; cryostorage).

6)     Production of a stable gene-expressing cell line (packaging of vector DNA; transduction of mammalian cells; selection).

7)     Production of a clonal gene-expressing cell line (packaging of vector DNA; transduction of mammalian cells; selection; limiting dilution cloning; expansion of [~25] clones).

8)    Characterization of stable gene-expressing cell line (analysis of cell line for trans gene expression by FACS or immunofluorescence, etc.).

Bacterial Unit (Dr. Elson)

                                                                                                           

9)       Analysis of bacterial flora via 16S ribosomal DNA for validation of monocolonization or germ-free status (PCR and agarose gel; PCR and DGGE; Elution of bands, cloning, and sequencing).

10)    Collection, 16s RNA characterization and cryopreservation of  bacteria. 

11)    Cloning of genes for bacterial expression (PCR, cloning, screening, sequencing). 

12) Expression and provision of bacterial protein relevant to inflammatory bowel disease, per protein.