Studies in transgenic mice: Insertional mutation at thepcd locus and absent de novo DNA methylation in trophoblast.
Item
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Title
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Studies in transgenic mice: Insertional mutation at thepcd locus and absent de novo DNA methylation in trophoblast.
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Identifier
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AAI9009751
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identifier
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9009751
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Creator
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Krulewski, Thomas Frank.
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Contributor
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Adviser: Jon W. Gordon
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Date
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1989
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Language
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English
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Publisher
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City University of New York.
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Subject
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Biology, Genetics | Biology, Molecular
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Abstract
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Two projects involving transgenic mice were undertaken: The characterization of an insertional mutation discovered in one line of animals, and the utilization of the foreign gene insert to report the effects of germ line transmission upon DNA methylation.;In the first project 17 lines of transgenic mice produced by pronuclear microinjection with a recombinant plasmid encoding a dihydrofolate reductase cDNA. In pedigree P432 one-quarter of pups derived from crosses of hemizygotes developed severed ataxias at about postnatal day 20. Southern hybridization intensities showed that ataxic animals were homozygous for the foreign gene insert. Extensive histologic investigations demonstrated the degeneration of cerebellar Purkinje cells, retinal photoreceptor cells and olfactory bulb mitral cells, as well as abnormalities of spermatogenesis. Analyses of Giemsa-banded metaphase-arrested chromosomes of transgenic fibroblasts revealed no gross aberrations. When carrier transgenic mice were bred to mice with the spontaneous mutation Purkinje cell degeneration (pcd) mutant pups were among the litters, thereby establishing allelism. DNA from transgenic mutants was used to construct a recombinant bacteriophage library which was screened for homology with the foreign gene insert. Two positive recombinant clones were identified as containing flanking mouse genomic DNA. Subfragments of these clones may be used to screen for RNA transcripts of the pcd gene. The cloning of pcd will allow for molecular analyses of a gene important to the regulation of neuronal development.;In the second project, de novo methylation in the absence of parental gene imprinting was studied in five transgenic pedigrees produced by pronuclear microinjection of linearized pBR322 sequences and delivered by Cesaerian section on day 19 of gestation. Placental DNA from founder mice was shown to be strikingly hypomethylated, and perhaps methylation free. Germline transmission was associated with increased placental DNA methylation. These results demonstrate a unique pattern of DNA processing in the placental (trophoblast) lineage, characterized by low or absent de novo methylation of unimprinted foreign DNA.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
