PMC:1540739 / 1130-6126 JSON TXT

Annnotations TAB JSON ListView MergeView

craft-sa-dev

craft-ca-core-ex-dev

craft-ca-core-dev

2_test

{"project":"2_test","denotations":[{"id":"16870721-12909629-76660890","span":{"begin":767,"end":768},"obj":"12909629"},{"id":"16870721-15456863-76660890","span":{"begin":767,"end":768},"obj":"15456863"},{"id":"16870721-14729946-76660890","span":{"begin":767,"end":768},"obj":"14729946"},{"id":"16870721-16006521-76660890","span":{"begin":767,"end":768},"obj":"16006521"},{"id":"16870721-16616333-76660890","span":{"begin":767,"end":768},"obj":"16616333"},{"id":"16870721-12074888-76660891","span":{"begin":1954,"end":1955},"obj":"12074888"},{"id":"16870721-9345015-76660892","span":{"begin":2856,"end":2857},"obj":"9345015"},{"id":"16870721-2783482-76660893","span":{"begin":3111,"end":3112},"obj":"2783482"},{"id":"16870721-9345015-76660894","span":{"begin":3374,"end":3375},"obj":"9345015"},{"id":"16870721-10525404-76660895","span":{"begin":3376,"end":3377},"obj":"10525404"},{"id":"16870721-11237328-76660895","span":{"begin":3376,"end":3377},"obj":"11237328"},{"id":"16870721-12136089-76660895","span":{"begin":3376,"end":3377},"obj":"12136089"},{"id":"16870721-16028230-76660895","span":{"begin":3376,"end":3377},"obj":"16028230"},{"id":"16870721-11584291-76660896","span":{"begin":3480,"end":3482},"obj":"11584291"},{"id":"16870721-16204450-76660897","span":{"begin":3878,"end":3880},"obj":"16204450"},{"id":"16870721-12563279-76660898","span":{"begin":4097,"end":4099},"obj":"12563279"},{"id":"16870721-14994271-76660898","span":{"begin":4097,"end":4099},"obj":"14994271"},{"id":"16870721-16382151-76660898","span":{"begin":4097,"end":4099},"obj":"16382151"},{"id":"16870721-16028230-76660899","span":{"begin":4169,"end":4171},"obj":"16028230"},{"id":"16870721-14994271-76660900","span":{"begin":4408,"end":4410},"obj":"14994271"}],"text":"INTRODUCTION\np53 is one of the most highly analyzed proteins for the past 25 years. Studies in cultured cells, often relying on the transfection of plasmids expressing various p53 mutants, have established models to explain how p53 is regulated. In recent years, some of these models were tested in vivo by targeting subtle mutations at the p53 locus using homologous recombination in embryonic stem (ES) cells to generate mutant mice. The strength of this approach is that mutations are tested in a genomic setting and expressed from the endogenous promoter, ensuring physiological expression levels and correct spatio-temporal profiles. As significant differences between phenotypes from targeted p53 mutants in vivo and transfection data were observed [e.g. Refs (1–5)], more targeted mutations need to be generated and analyzed in multiple tissues to formulate more accurate models of p53 regulation.\nHowever, using homologous recombination in ES cells to generate mutant mice is an inefficient, slow and expensive method because (i) homologous recombination typically occurs at low frequency in ES cells, requiring sophisticated selection schemes and screening of hundreds of clones to identify the desired mutant; (ii) large (15–20 kb) plasmids, often difficult to clone, are required to increase targeting efficiency and (iii) breeding mice to homozygosity and housing a mouse colony generate further delays and costs. Such limitations make the repeated targeting of a locus a technically daunting and economically impractical task.\nImprovements in current technologies are needed to enable such analyses to be applied to the p53 or other genes. Developing methods to increase targeting efficiency in ES cells is clearly an important goal. In addition, efficient methods for gene targeting in fibroblasts could expedite phenotypic analyses. Indeed, siRNAs in fibroblasts often provide a faster read-out than equivalent gene knock-outs in animals (6). However, modeling most disease-associated mutations requires generating subtle mutations, not knock-outs or reduced expression alleles. Targeting point mutations in fibroblasts by homologous recombination is extremely inefficient, and targeting both alleles is required to reveal the phenotype of recessive autosomal mutations.\nHere we report an approach that enables highly efficient targeting at the p53 locus in both ES cells and fibroblasts. Recombinase-Mediated Cassette Exchange (RMCE) approaches were developed to improve targeting efficiency using a two-step process: the gene of interest is first replaced by a selection cassette flanked by recombinase target sites (e.g. loxP sites for Cre recombinase, to create a ‘floxed’ locus). Then, Cre-mediated recombination in the presence of a cassette containing a floxed mutant allele removes the resident sequence and inserts the mutant gene (7). Previously, technical difficulties have prevented RMCE from being applied routinely to generate mutant mice. For example, exchanges using cassettes with directly repeated loxP sites were inefficient because excisions dominated the intended exchanges (8). loxP sites with different sequences were generated to overcome this problem, but these sites also underwent intramolecular recombination, making RMCE efficient only if the replacement cassette contained a marker enabling selection of the desired recombinant (7,9–12). However, interference resulting from expression of the selection marker and the endogenous gene (13) necessitates strategies to remove the selectable gene. Together, previous studies indicate that an optimal RMCE requires (i) inverted heterologous loxP sites diverging by at least 2 nt to maximize the efficiency of exchange and (ii) an expression cassette enabling both positive selection to identify the initial recombinant and then negative selection to obtain a ‘marker-free’ mutant allele (14).\nMost RMCE experiments have been performed at random sites in somatic cell lines. Only a few mutant mice generated by RMCE in ES cells have been reported, but the RMCE systematically introduced a selectable marker (15–17), or, when tested without an incoming marker, proved inefficient (12). A recent report disclosed an additional problem: the Hygromycin–Thymidine Kinase fusion gene used most frequently for positive/negative selection in RMCE, leads to mouse sterility, so that exchanges can only be performed in ES cells (16).\nThe RMCE strategy presented here relies on the integrated use of inverted heterologous loxP sites, a positive/negative selection marker that preserves the germline capacity of ES cells, and the power of mouse genetics to expedite phenotypic analyses. We show that our approach enables efficient targeting of marker-free mutations at the p53 locus in ES cells to generate mutant mice, but more importantly, it is Adapted for targeting in Somatic cells to Accelerate Phenotyping (ASAP). Because it relies on very general principles, RMCE-ASAP could be applied to any locus of interest."}

PMC:1540739 / 1130-6126 JSONTXT

Annnotations TAB JSON ListView MergeView

craft-sa-dev

craft-ca-core-ex-dev

craft-ca-core-dev

2_test

PMC:1540739 / 1130-6126 JSON TXT