Targeted disruption of the murine int-1 proto-oncogene resulting in severe abnormalities in midbrain and cerebellar development.

Journal ArticleThe int-1 proto-oncogene was first identified as a gene activated in virally induced mouse mammary tumours. Expression studies, however, suggest that the normal function of this gene may be in spermatogenesis and in the development of the central nervous system. Genes sharing sequence similarity with int-1 have been found throughout the animal kingdom. For example, int-1 has 54% amino-acid identity to the Drosophila segment polarity gene wingless (wg). Both the int-1 and wg gene products seem to be secreted proteins, presumably involved in cell-cell signalling. We have now explored the function of int-1 in the mouse by disrupting one of the two int-1 alleles in mouse embryo-derived stem cells using positive-negative selection. This cell line was used to generate a chimaeric mouse that transmitted the mutant allele to its progeny. Mice heterozygous for the int-1 null mutation are normal and fertile, whereas mice homozygous for the mutation may exhibit a range of phenotypes from death before birth to survival with severe ataxia. The latter pathology in mice and humans is often associated with defects in the cerebellum. Examination of int-1-/int-1- mice at several stages of embryogenesis revealed severe abnormalities in the development of the mesencephalon and metencephalon indicating a prominent role for the int-1 protein is in the induction of the mesencephalon and cerebellum

Introduction of homologous DNA sequences into mammalian cells induces mutations in the cognate gene.

Journal ArticleInjection of homologous DNA sequences into nuclei of cultured mammalian cells induces mutations in the cognate chromosomal gene. It appears that these mutations result from incorrect repair of a heteroduplex formed between the introduced and the chromosomal sequence. This phenomenon is termed 'heteroduplex induced mutagenesis'. The high frequency of these events suggests that this method may prove useful for introducing mutations into specific mammalian genes

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Efficient correction of mismatched bases in plasmid heteroduplexes injected into cultured mammalian cell nuclei.

Journal ArticleHeteroduplexes were prepared from two plasmids, pRH4-14/TK and pRH5-8/TK, containing different amber mutations in the neomycin resistance gene (Neor). The Neor gene was engineered to be expressed in both bacterial and mammalian cells. A functional Neor gene conferred kanamycin resistance to bacteria and resistance to the drug G418 to mammalian cells. In addition, the plasmids contained restriction site polymorphisms which did not confer a selectable phenotype but were used to follow the pattern of correction of mismatched bases in the heteroduplexes. In a direct comparison of the efficiency of transforming mouse LMtk- cells to G418r, the injection of heteroduplexes of pRH4-14/TK-pRH5-8/TK was 10-fold more efficient than the coinjection of pRH4-14/TK and pRH5-8/TK linear plasmid DNA. In fact, injection of 5 to 10 molecules of heteroduplex DNA per cell was as efficient in transforming LMtk- cells to G418r as the injection of 5 to 10 molecules of linear plasmid DNA per cell containing a wild-type Neor gene. To determine the pattern of mismatch repair of the injected heteroduplexes, plasmids were "rescued" from the G418r cell lines. From this analysis we conclude that the generation of wild-type Neor genes from heteroduplex DNA proceeds directly by correction of the mismatched bases, rather than by alternative mechanisms such as recombination between the injected heteroduplexes. Our finding that a cell can efficiently correct mismatched bases when confronted with preformed heteroduplexes suggests that this experimental protocol could be used to study a wide range of DNA repair mechanisms in cultured mammalian cells

Nonreciprocal exchanges of information between DNA duplexes coinjected into mammalian. cell nuclei

Journal ArticleWe have examined the mechanism of homologous recombination between plasmid molecules coinjected into cultured mammalian cells. Cell lines containing recombinant DNA molecules were obtained by selecting for the reconstruction of a functional Neor gene from two plasmids that bear different amber mutations in the Neor gene. In addition, these plasmids contain restriction-length polymorphisms within and near the Neor gene. These polymorphisms did not confer a selectable phenotype but were used to identify and categorize selected and nonselected recombinant DNA molecules. The striking conclusion from this analysis is that the predominant mechanism for the exchange of information between coinjected plasmid molecules over short distances (i.e., less than 1 kilobase) proceeds via nonreciprocal homologous recombination. The frequency of homologous recombination between coinjected plasmid molecules in cultured mammalian cells is extremely high, approaching unity. We demonstrate that this high frequency requires neither a high input of plasmid molecules per cell nor a localized high concentration of plasmid DNA within the nucleus. Thus, it appears that plasmid molecules, once introduced into the nucleus, have no difficulty seeking each other out and participating in homologous recombination even in the presence of a vast excess of host DNA sequences. Finally, we show that most of the homologous recombination events occur within a 1-h interval after the introduction of plasmid DNA into the cell nucleus

High-fidelity gene targeting in embryonic stem cells by using sequence replacement vectors.

Journal ArticleMutations were targeted to the Hprt locus in murine embryonic stem cells by using sequence replacement vectors. When the vector was designed such that the mutated sequences were flanked on both sides by several kilobases of DNA homologous to the target locus, replacement of chromosomal sequences with the exogenous DNA occurred with precision. If, on the other hand, the target-homologous DNA on one arm of the vector was reduced to below 1 kb in length, the fidelity of recombination was diminished

Location of crossovers during gene targeting with insertion and replacement vectors.

Journal ArticleGene targeting was used to introduce nonselectable genetic changes into chromosomal loci in mouse embryo-derived stem cells. The nonselectable markers were linked to a selectable marker in both insertion- and replacement-type vectors, and the transfer of the two elements to the Hprt locus was assayed. When insertion vectors were used as substrates, the frequency of transfer was highly dependent upon the distance between the nonselectable marker and the double-strand break in the vector. A marker located close to the vector ends was frequently lost, suggesting that a double-strand gap repair activity is involved in vector integration. When replacement vectors were used, cotransfer of a selectable marker and a nonselectable marker 3 kb apart was over 50%, suggesting that recombination between vector and target often occurs near the ends of the vector. To illustrate the use of replacement vectors to transfer specific mutations to the genome, we describe targeting of the delta F508 mutation to the CFTR gene in mouse embryo-derived stem cells

Aortic dilatation after endovascular repair of blunt traumatic thoracic aortic injuries

ObjectiveEndovascular repair of blunt traumatic thoracic aortic injuries (BTAI) has become routine at many trauma centers despite concerns regarding durability and aortic dilatation in these predominantly young patients. These concerns prompted this examination of thoracic aortic expansion after endovascular repair of a BTAI.MethodsThe immediate postoperative and most recent computed tomography (CT) scans of patients who had undergone urgent endovascular repair of a BTAI and had at least 1 year of follow-up were reviewed. Diameter measurements were made at four predetermined sites: immediately proximal to the left subclavian artery (D1), immediately distal to the left subclavian artery (D2), distal extent of the endograft (D3), and 15 mm beyond the distal end of the endograft (D4). Split screens permitted direct comparison of measurements between CTs at the corresponding levels.ResultsDuring a 6-year period (2001-2007), 21 patients (mean age, 42.9 years; range, 19-81 years) underwent endovascular repair of a BTAI, 17 with at least 1 year of follow-up (mean, 2.6 years; range, 1-5.5 years). No patients required reintervention during this period. The mean rate of dilatation for each level of the thoracic aorta in mm/year was: D1, 0.74 (95% confidence interval [CI], 0.42-1.06); D2, 0.83 (95% CI, 0.55-1.11); D3, 0.63 (95% CI, 0.37-0.89); D4, 0.47 (95% CI, 0.27-0.67). The rate of expansion of D2 differed significantly vs D4 (P = .025).ConclusionsDuring the first several years of follow-up, the proximal thoracic aorta dilates minimally after endovascular repair of BTAIs, with the segment just distal to the left subclavian artery expanding at a slightly greater rate. Longer-term follow-up is necessary to determine whether this expansion continues and becomes clinically significant

Cytochrome P450 2B diversity and dietary novelty in the herbivorous, desert woodrat (Neotoma lepida)

pre-printDetoxification enzymes play a key role in plant-herbivore interactions, contributing to the on-going evolution of ecosystem functional diversity. Mammalian detoxification systems have been well studied by the medical and pharmacological industries to understand human drug metabolism; however, little is known of the mechanisms employed by wild herbivores to metabolize toxic plant secondary compounds. Using a wild rodent herbivore, the desert woodrat (Neotoma lepida), we investigated genomic structural variation, sequence variability, and expression patterns in a multigene subfamily involved in xenobiotic metabolism, cytochrome P450 2B (CYP2B). We hypothesized that differences in CYP2B expression and sequence diversity could explain differential abilities of woodrat populations to consume native plant toxins. Woodrats from two distinct populations were fed diets supplemented with either juniper (Juniperus osteosperma) or creosote bush (Larrea tridentata), plants consumed by woodrats in their respective desert habitats. We used Southern blot and quantitative PCR to determine that the genomic copy number of CYP2B in both populations was equivalent, and similar in number to known rodent copy number. We compared CYP2B expression patterns and sequence diversity using cloned hepatic CYP2B cDNA. The resulting sequences were very diverse, and clustered into four major clades by amino acid similarity. Sequences from the experimental treatments were distributed non-randomly across a CYP2B tree, indicating unique expression patterns from woodrats on different diets and from different habitats. Furthermore, within each major CYP2B clade, sequences shared a unique combination of amino acid residues at 13 sites throughout the protein known to be important for CYP2B enzyme function, implying differences in the function of each major CYP2B variant. This work is the most comprehensive investigation of the genetic diversity of a detoxification enzyme subfamily in a wild mammalian herbivore, and contributes an initial genetic framework to our understanding of how a wild herbivore responds to critical changes in its diet