The overall objectives of this research are to describe and understand the chromosomal changes which may be occurring in the Raga pipiens karyotype during normal embryogenesis, the development of neoplasia and the temperature the chromosomal incorporation site(s) of the Lucke Tumor Herpesvirus (LTV) genome. The genomes of embryonic cells, some highly differentiated cells, and Lucke' tumor cells can be """"""""reprogrammed"""""""" by nuclear transplant to develop along pluripotent pathways. These unusual properties of Raga pipiens and its associated Lucke' tumor make it an excellent system for analysis of the chromosomal modifications, and their stability, occurring during normal and neoplastic development. An understanding of such modifications will enhance our knowledge of how a common, although pluripotent, genome can give rise to the diversity of cellular phenotypes observed in the vertebrate organism. This goal will be achieved by attainment of four specific aims:(1) continuation of the current efforts to describe the banded karyotype of PNKT-4B and Lucke' tumor cells, (2) development in this laboratory of the capability to propagate the Lucke' tumor, (3) examination of the stability of normal and neoplastic karyotype during """"""""reprogrammed"""""""" embryonic development following nuclear transplant, and (4) determination of the chromosomal site(s) of LTV genome incorporation by FISH. Chromosomal loci of NORs, N-bands, are visualized by staining with acidic colloidal silver nitrate and C-bands are induced y hot alkali treatment and Giemsa staining. Elongated prometaphase chromosomes from MTX synchronized cultures can be G-banded by mild trypsin digestion and Giemsa staining. R-bands (replication) and SCEs can be visualized by the incorporation of BrdU into replicating chromosomes followed by differential Giemsa staining. Accomplishment of these specific aims will provide evidence on the role of chromosomal changes involved in the development, long term existence, expression of the metastatic phenotype and developmental potential of Lucke' tumor and normal embryonic cells. Finally, this project will provide a significant opportunity for students to gain hands on experience in biomedical research and should act as a motivating factor encouraging their entry into a research oriented career.
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