RESUMO
The Escherichia coli bacteriophage P1 packages host chromosome separately from phage DNA, and transfers it to recipient cells at low frequency in a process called generalized transduction. Phage genomes are packaged from concatemers beginning at a specific site, pac. To increase transduction rate, we have inserted pac into the chromosome at up to five equally spaced positions; at least this many are fully tolerated in the absence of P1 infection. A single chromosomal pac greatly increases transduction of downstream markers without decreasing phage yields; 3.5 × as much total chromosomal DNA is packaged. Additional insertions decrease phage yield by > 90% and also decrease phage DNA synthesis, although less dramatically. Packaging of chromosomal markers near to and downstream of each inserted pac site is, at the same time, increased by greater than 10 fold. Transduction of markers near an inserted pac site can be increased by over 1000-fold, potentially allowing identification of such transductants by screening.
Assuntos
Bacteriófago P1/fisiologia , Cromossomos Artificiais de Bacteriófago P1/fisiologia , Cromossomos Bacterianos/genética , DNA Bacteriano/genética , Escherichia coli/genética , Cromossomos Artificiais de Bacteriófago P1/genética , DNA Viral/análise , DNA Viral/fisiologia , Hibridização de Ácido Nucleico , Transdução GenéticaRESUMO
The ability to restrict gene expression or disruption to specific regions of the brain would enhance understanding of the molecular basis for brain development and function. For this purpose, brain region-restricted promoters are essential. Here we report the isolation of a DNA fragment containing the Emx1 gene promoter, which is responsible for dorsal telencephalon-specific expression. The Cre recombinase gene was inserted into a mouse PAC (P1-derived artificial chromosome) Emx1-locus clone (PAC-Emx1#1 clone) and utilized to generate three transgenic mouse lines. In all three lines, especially Tg3, Cre-mediated recombination was highly restricted to Emx1-expressing cell lineages, from embryonic stages to adulthood. Immunohistochemical analyses showed that Cre protein is expressed in the dorsal telencephalon in all three lines in adulthood. Thus, the PAC-Emx1#1 clone contains essentially all regulatory elements necessary for Emx1 gene expression. Our results suggest that Emx1-Cre Tg3 mice and the PAC-Emx1#1 clone constitute powerful tools for dorsal telencephalon-specific gene manipulation.
Assuntos
Cromossomos Artificiais de Bacteriófago P1/fisiologia , Proteínas de Homeodomínio/genética , Integrases/genética , Integrases/metabolismo , Camundongos Transgênicos/genética , Recombinação Genética , Telencéfalo/fisiologia , Animais , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Óperon Lac , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos/embriologia , Camundongos Transgênicos/crescimento & desenvolvimento , Regiões Promotoras Genéticas , Fatores de TranscriçãoRESUMO
Loss-of-function approaches by the Cre/loxP technology have provided powerful tools for functional analyses of genes of interest expressed preferentially in a particular tissue. Here we describe the generation of transgenic mouse lines expressing Cre recombinase under the control of the promoter/enhancer unit of the gene for the alpha2 chain of collagen type I (Col1alpha2). As an expression vector, we used a P1-derived artificial chromosome (PAC), which harbors approximately 100 kb carrying the col1alpha2 gene. The improved coding sequence of the Cre recombinase was introduced to replace the first exon of col1alpha2. Cre expression was determined by immunohistochemistry and Cre-mediated onset of beta-galactosidase expression in ROSA26R-Cre reporter mice. In four analyzed transgenic lines, Cre recombinase was efficiently expressed during embryogenesis and in adult animals in cells of mesenchymal origin, such as dermal fibroblasts, mesenchymal cells of blood vessel walls, and cells in fibrous connective tissues surrounding internal organs.
