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Title: The development of a large interval recombinase mediated cassette exchange (RMCE) strategy
Author: Penfold, Catherine
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2005
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Murine embryonic stem (ES) cells have provided researchers with a useful tool to investigate genome function and the consequences of genome mutation. One mutational approach is gene-targeting, this involves the introduction of DNA sequences of choice, precisely, to almost any location in the target genome by homologous recombination. At present, most gene-targeting strategies introduce DNA constructs that derive from plasmids. Plasmids can stably propagate up to approximately 30 kb of DNA. Therefore, this size limit may place a restriction on the range of mutations that may be made to a genome using a single plasmid-derived gene-targeting construct alone. To overcome this limitation, multiple rounds of sequential gene-targeting experiments may be performed, however such an approach may be too lengthy to be practicable. In order to address this current limitation with gene-targeting a novel strategy was tested, implementing Cre-lox site-specific recombination (SSR) technology and the bacterial artificial chromosome (BAC) vector system. Two sequential gene-targeting events in murine E14Tg2a ES cells (HPRT) were performed at separate locations to chromosome 11. The aim of gene-targeting was to create an interval on chromosome 11 that included a single copy of the murine alpha-globin locus, between the hetero-specific lox sites, loxP and lox511, an interval of approximately 64 kb. To this end the first targeting event delivered lox511 /hygromycin/I See Illox51 J sequences and the second event frt/I See I/5'hprt//oxP/neomycin sequences. ES cells that were confirmed to have correctly undergone the two desired targeting events (double-targeted) were then assessed to determine whether these events had occurred to the same chromosome 11 (in eis ), as desired, or to the alternate copies of chromosome 11 (in trans). This assessment involved restricting DNA from the double-targeted ES cell lines with the rare-cutting restriction endonuclease I See I and resolving the products of this restriction by pulsed field gel electrophoresis. This analysis identified two in cis lines (CAT-A3 and CAT-B3) and an in trans line (CATCIO). The double-targeted ES cell lines were then further characterised to determine whether the hetero-specific lox sites they harboured would participate in ere-mediated SSR. The positive result of this analysis was the generation of ES cell clones that were hemizygous for the alpha-globin locus, a deletion of 64 kb. Hemizygous ES cell clones were obtained from the CAT-A3 and CAT-B3 ES cell lines, as predicted, but not from the CAT-C 10 line, although all the lines tested showed evidence of SSR occurring. In parallel to achieving the interval between loxP and lox51 l in ES cells, a BAC, harbouring the alpha-globin locus, was similarly modified with lox sites using recombination-mediated cloning. The aim of the BAC modification was to create an interval between lox sites in the BAC identical to that achieved in the ES cells. The BAC was targeted sequentially with two separate constructs, lox511/k.anamycin/lox511/HSVtk and then blasticidin/loxP/3'hprt/I See 11.frt. The correct targeting of the BAC was verified by restricting its DNA with a panel restriction endonucleases. The lox sites were then tested in an in vitro analysis with purified Cre recombinase and found to be competent to participate in SSR reactions. The modified BAC was co-electroporated with a Cre expression plasmid into the CAT-A3 and CAT-B3 ES cell lines, previously characterised as targeted in eis, with the aim of exchanging the interval sequences in the ES cell with those of the BAC. The ultimate aim of such an exchange would be to deliver any combination of mutations that would be previously engineered to the BAC interval, to that of the ES cell, by a single SSR event. This experimental approach should expedite and facilitate the mutational analysis of gene loci. To generate comparative data the result of SSR between the modified BAC and an in trans targeted ES cell line (CAT-CI 0) was also assessed. The selection for the desired exchange involved reconstruction of an Hprt minigene and exclusion of a thymidine kinase gene, cells which haboured these events could therefore be selected for in HAT and ganciclovir supplemented media respectively. ES cell clones generated from both of the in cis lines tested (CAT-A3 and CAT-B3) had the correct selection resistance profiles, thus indicating that the desired exchange had been achieved in these clones. Additionally, Southern blot analysis from the DNA from these clones was consistent with the achievement of the desired exchange. However, the results obtained from clones generated from the in trans line (CAT-CI 0) were not consistent with their predicted genetic arrangement following SSR with the modified BAC. Thus far similar experimental approaches have been implemented to exchange smaller intervals of I to 5 kb and have been termed recombinase mediated cassette exchange (RMCE). However the experiments described within this thesis are the first test whether the same rationale may be applied to larger intervals. The strategy described and tested in this thesis has therefore been termed large interval RMCE (liRMCE).
Supervisor: Medvinsky, Alexander ; Smith, Andrew Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Genome function ; Recombination, Genetic