Cloning and analysis of mouse chromosomal loci specifically active in embryonal carcinoma stem cells

Bhat, Krishna Moorthi (1989) Cloning and analysis of mouse chromosomal loci specifically active in embryonal carcinoma stem cells. Doctoral (PhD) thesis, Memorial University of Newfoundland.

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    Available under License - The author retains copyright ownership and moral rights in this thesis. Neither the thesis nor substantial extracts from it may be printed or otherwise reproduced without the author's permission.
    (Original Version)

Abstract

Chromosomal loci that are specifically active in the mouse embryonal carcinoma stem cells were cloned by using a functional selection procedure. The pluripotent P19 embryonal carcinoma cells were transfected with an enhancer-trap plasmid containing an enhancerless, inactive neomycin resistance gene and NEO⁺-transformant cell lines were isolated. When the cells were induced to differentiate, most of the cell lines continued to express the neomycin resistance gene, however, in some cell lines, the neomycin resistance gene because repressed. From the later group of cell lines, eight in total, the integrated transgene plus the flanking cellular DNA sequences were cloned. Three of the cloned fragments from the above eight cell lines possessed a high NEO⁺-transforming enhancer activity in the undifferentiated P19 cells. Among these three, two were inactive in differentiated P19 cells in NIH 3T3 cells, while the remaining one was active in both these differentiated cell types. Further analysis of these stem cell specific enhancers revealed that they were derived from the stem-cell specific Early Transposon-like genes. -- In order to search for the presence of genes in the above stem cell specific loci, a P19 genomic library was constructed and the preinsertion regions at the neomycin resistance gene-integration sites were cloned from these cell lines. The cloned DNA was analyzed for the presence of genes by Northern blotting analysis. Messages were detected in the Northern blots against some of the loci, however, their identity as functional genes is yet to be established. -- During the course of this investigation, I observed the presence of Early Transposon-like genes in three of the above loci. Restriction mapping of the preinsertion loci and the Southern blot analysis of the DNA from mouse testis, parent P19 cells, and the three NEO⁺ cell lines with the locus-specific probes, provided direct evidence that the transposon was inserted into these loci during the experimental time-frame and therefore was movable in the mouse genome. Analysis of the cell extracts from the three embryonal carcinoma cell lines, P19, F9, and PCC3 with a transposon-specific probe detected extrachromosomal copies of this transposon only in the P19 cells. Southern blot analysis of the DNA from mouse germ cell and various somatic cell lineages with the ends-specific transposon probes indicated that there were no apparent differences in the transposon integration sites between the germ line and the soma, suggesting that transposition of these ETn-like genes is strictly stem cell specific and ceases to occur before allocation of founder cells to the germ cell lineage and somatic lineages during mouse embryogenesis. These results demonstrate that the early transposon-like genes can act as a powerful insertion mutagen in the founder cells of the mouse embryo.

Item Type: Thesis (Doctoral (PhD))
URI: http://research.library.mun.ca/id/eprint/1199
Item ID: 1199
Additional Information: Bibliography: leaves 164-181.
Department(s): Medicine, Faculty of
Date: 1989
Date Type: Submission
Library of Congress Subject Heading: Embryology--Mammals; Mice; Molecular cloning
Medical Subject Heading: Cloning, Molecular; Stem Cells; Mammals--Embryology; Carcinoma, Embryonal

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