We have produced 17 lines of transgenic mice by microinjecting a gene coding for an altered dihydrofolate reductase. The gene is cloned as a cDNA inserted into an expression vector. We have thus far made three observations which form the basis of this proposal. First, we have found that when digested with the restriction enzymes SalI and PstI, the plasmid integrates as a single unit per host genome with high frequency. Because it contains no restriction sites for BamHI or EcoRI, this fragment can be rapidly cloned from the genomes of transgenic mice along with both 5' and 3' flanking mouse DNA. Second, expression of the cDNA in heterozygous transgenic mice is associated with a variety of developmental abnormalities. Third, a recessive insertional mutant in one of the established pedigrees appears as a severe neurologic disturbance beginning at 19 days of postnatal development. These findings will be used in the following three ways to analyze mouse development: A non-coding fragment with SalI and PstI ends will be introduced into embryos and the resultant transgenic mice will be bred to homozygosity for detection of insertional mutagenesis. Then, exploiting the unique characteristics of this fragment, we will rapidly clone and analyze the affected host genes. The abnormalities in heterozygous animals expressing the cDNA will also be analyzed. We will trace the cell lineages affected in the animals using in situ hybridization techniques that can distinguish foreign from host DHFR gene expression. Sequential developmental studies of this type will be carried out in order to trace the affected cell lineages precisely and to determine at what point in ontogeny foreign gene expression within these lineages or in neighboring cells leads to the birth defects seen. Finally, we will produce and screen a bacteriophage library from our animals with the adult onset neurologic disorder and attempt to clone a mouse gene whose interruption has led to the neurologic disease. In this way we will retrieve and analyze a gene which appears to regulate adult development of the brain.
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