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Title: Human chromosome tagging in somatic cells, using SINE and LINE targeting vectors
Author: Watson, J. E. Vivienne
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1995
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A series of targeting vectors have been constructed containing two types of human-specific repeat sequences: Blur8 a short interspersed nuclear element (SINE) from the Alu family of sequences, and two fragments from the 3' end of a long interspersed element (LINE) L1. The vectors contain dominant markers selectable in mammalian cell culture (neoR), as well as in yeast (Leu2), and bacteria (Amp), and restriction sites to enable unique cleavage of genomic DNA after integration of the vector (ISce-I and lacO). I have introduced the vectors into J1c4, a somatic cell hybrid containing a single human chromosome in a background of Chinese hamster ovary (CHO) DNA. I have tested the ability of the chosen repeat sequences to mediate integration of the vector into human DNA in preference to Chinese hamster DNA. The sites of integration were localised by fluorescent in situ hybridisation (FISH). Forty-three G418-resistant clones were isolated after targeting with a vector containing two Alu-repeat sequences. Of 37 of these analysed by FISH, only in 1 had the vector integrated into the human chromosome 11. Using vectors containing two L1 fragments or an L1 and an Alu sequence, a total of 65 G418 resistant clones were isolated in three different experiments. FISH analysis showed that in 8/14 of these clones the marker was localised to the human chromosome 11. This suggests that fragment(s) from the L1 repetitive element can direct integration of a vector into human DNA within a somatic cell hybrid. Characterisation of clones containing markers in the human chromosome has been carried out using PCR and restriction endonuclease mapping strategies. Cleavage at a unique restriction endonuclease site introduced by the vector has been demonstrated.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available