Evidences in the literature indicate the existence of an embryonic insulin which could play a role in controlling fetal growth at stages when pancreas has not yet become functional. We have developed a mesodermal cell line called 1246-3A derived from C3H mouse teratoma which produces and secretes in culture a growth factor called insulin-related factor (IRF) which is immunologically and biochemically similar to pancreatic insulin. IRF can be purified to homogeneity from 1246-3A conditioned medium in a very rapid, efficient procedure which can be easily scaled up. It includes CG50 chromatography, immunoaffinity chromatography and reverse phase HPLC. As pure IRF can be obtained in Mug quantity it will be possible to determine its structure and study the regulation of its secretion and its expression in vitro and in vivo. These experiments are performed in order to investigate the possible involvement of IRF as a growth factor during the pre-pancreatic stage of embryonic development. More specifically, IRF amino acid sequence will be determined by gas phase sequencing. Based on this data, oligonucleotide sequences corresponding to IRF sequence will be synthesized and used to clone cDNA encoding for IRF from a 1246-3A cDNA library, in order to investigate the regulation of the gene coding for IRF during embryonic development. Partial cDNA sequence analysis will be performed in order to compare it to the pancreatic insulin gene. This system presented in this proposal should prove a suitable one to the long-term goal of investigating the nature and function of emryonic extra-pancreatic insulin produced by mesodermal tissue and to understand its function in vivo as a growth factor during development.
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