Abstract
BackgroundChromatin adjoining the site of integration of a transgene affects expression and renders comparisons of closely related transgenes, such as those derived from a BAC deletion series retrofitted with enhancer-traps, unreliable. Gene targeting to a pre-determined site on the chromosome is likely to alleviate the problem.FindingsA general procedure to replace the loxP site located at one end of genomic DNA inserts in BACs with lox66 is described. Truncating insert DNA from the loxP end with a Tn10 transposon carrying a lox66 site simultaneously substitutes the loxP with a lox66 sequence. The replacement occurs with high stringency, and the procedure should be applicable to all BACs in the public domain. Cre recombination of loxP with lox66 or lox71 was found to be as efficient as another loxP site during phage P1 transduction of small plasmids containing those sites. However the end-deletion of insert DNA in BACs using a lox66 transposon occurred at no more than 20% the efficiency observed with a loxP transposon. Differences in the ability of Cre protein available at different stages of the P1 life cycle to recombine identical versus non-identical lox-sites is likely responsible for this discrepancy. A possible mechanism to explain these findings is discussed.ConclusionsThe loxP/lox66 replacement procedure should allow targeting BACs to a pre-positioned lox71 site in zebrafish chromosomes; a system where homologous recombination-mediated "knock-in" technology is unavailable.
Highlights
Chromatin adjoining the site of integration of a transgene affects expression and renders comparisons of closely related transgenes, such as those derived from a bacterial artificial chromosome (BAC) deletion series retrofitted with enhancer-traps, unreliable
Research Hypothesis Expression of a transgene integrated into the germline of an animal is influenced by i) variation in copy number of transgene and ii) the effect of chromatin adjoining the site of integration [1]
Progressive end-deletions using the lox66 transposons were generated at an overall efficiency of 10-20% of that obtained with the wild type loxP transposons, depending on whether other recombinogenic sites existed in the genomic DNA insert of the BAC clone
Summary
Research Hypothesis Expression of a transgene integrated into the germline of an animal is influenced by i) variation in copy number of transgene and ii) the effect of chromatin adjoining the site of integration [1]. Expression comparisons of closely related transgenes, such as those derived from a BAC deletion series retrofitted with enhancer-traps [2], become unreliable. Targeting transgenes to the same site on the chromosome alleviates the problem, and both “knock-in” technology using homologous recombination [1] and insertion of cDNA plasmids into a loxP site have been used in previous studies [3]. Because the lox-Cre recombination reaction/equilibrium favors excision, mutant sites such as lox and lox have been constructed to stably incorporate small plasmid DNA into chromosomes of plants and mouse ES cells [4,5]. BACs have not been used presumably because altering the loxP or lox511 sites flanking insert DNA in BACs is challenging.
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