GENE TARGETING OF THE MOUSE ONCOSTATIN M GENE IN EMBRYONIC STEM CELLS

Abstract

Oncostatin M (OSM) is an interleukin-6 (IL-6) -related cytokine, most closely related to the leukemia inhibitory factor (LIF) in secondary structure, genomic organization and gene size comparisons. The OSM cDNA is encoded by three exons with most of the coding region contained in the third exon. The protein precursor contains an amino terminal signal peptide, four alpha helices, and four highly charged proteolytic cleavage sites at the carboxyl terminus. OSM influences a wide variety of cells and causes a number of biological responses. The murine homolog (mOSM) of the human OSM (hOSM) gene has only recently been identified, and its isolation should allow extensive in vivo studies to determine its cellular function of this important cytokine. Studies of animals lacking a gene of interest can give important clues as to the normal function of that gene. While an in vivo approach in assessing gene function may not be conclusive, a gene targeting ("Knock-Out") experiment is valuable in providing clues to gene function. This gene disruption approach has been used for several other members of the IL-6 cytokine family. Recent literature searches indicate that in vivo model systems have not been fully explored for the OSM gene. Therefore, a similar gene targeting experiment using the mOSM gene is a logical approach to study its function. This approach to studying the OSM gene may provide important information underlying the molecular mechanisms of normal cell growth and differentiation as well as oncogenesis. Further characterization of this gene is essential to understand its specific function. The overall objective of this project is to construct a gene targeting vector and demonstrate its utility in embryonic stem(ES) cells for use in the subsequent creation of mOSM gene-deficient mice. In general, in gene targeting ("Knockout") experiments, ES cells are genetically altered and the resultant cells can be transmitted to the germline producing embryo chimeras. The mOSM gene targeting vector was designed to mutate the endogenous mOSM gene by replacement of the coding region of exon III, with a positive selectable marker (neomycin-resistance) gene. The exon III region of the mOSM gene was chosen since it includes the receptor-binding domains, and conserved cysteine residues that provide intramolecular disulfide bond linkages necessary to maintain tertiary structure, both of which are required for the biological activity of the OSM protein. The following strategy was used to produce mutant mOSM ES cell lines: (1) genomic clones that contain the mOSM gene were identified and isolated from a mouse genomic library; (2) each of the genomic clones were mapped using restriction enzyme analysis and Southern blot hybridizations to identify gene position and orientation; (3) the mapped clones were used in constructing a vector designed to target and mutate the endogenous mOSM gene; and (4) ES cells were transfected with the gene targeting vector and resulted in many clonal cell lines that were subsequently screened for homologous recombination events. In summary, the screening of the mouse genomic library resulted in the isolation of five genomic clones. Characterization of these clones resulted in the identification of genomic DNA insert size, and mOSM intron-exon organization. A 15.8 Kbp genomic clone was selected for use in constructing a gene targeting vector because it contained sufficient 5' and 3' genomic flanking DNA. Regions of both genomic flank arm sequences were mapped using several restriction enzymes. This process provided useful restriction enzyme data which was subsequently used to design the targeting vector. Portions of these genomic flank regions were used to engineer a gene targeting vector designed to mutate the endogenous mOSM gene in ES cells. Initial transfection experiments introducing the targeting vector into ES cells, resulted in the screening of 218 recombinant ES cell clones. Subsequent screening of these clones indicated that homologous recombination had not occurred. The frequency of a homologous recombination event in ES cells was quite variable and, therefore, more recombinant ES cell colonies need to be screened.