Fig. 1.
Fig. 1. Isolation and characterization of a murine PU.1 91-kb P1 genomic clone. / (A) Map of the genomic clone and strategy for insertion of the Thy1.1 reporter by homologous recombination. The top cartoon is a kilobase scale. The middle panel is a map of the murine PU.1 91-kb genomic insert (thick black line), with sequences from the P1 vector shown by the thinner black line. Shown are the relative locations of the 5 coding exons, labeled E1 to E5; restriction sites of enzymes used to map the clone; and the location of the murine equivalent of the 3′ end of the human tbp-1 gene at the extreme 5′ end of the insert. The bottom panel shows the strategy used for insertion of the Thy1.1 reporter by homologous recombination. “Genomic sequence” represents an expanded view of the murine PU.1 genomic region between exons 3 to 5. The panel below depicts the shuttle vector in which a 928-bp homologous fragment, including parts of exon 3, intron 3, and exon 4, were placed upstream of an IRES followed by the 544-bp murine Thy1.1 cDNA, a polyadenylation signal, and a 375-bp homologous portion of exon 5. “Homologous recombination” represents the final structure of the modified P1 vector, referred to as mPU.1-Thy1.1-P1 in the text. Following homologous recombination into the same region as was done for PU.1 knockout animals, we predict a genomic structure of the P1 as shown in the bottom figure in which a single transcript would encode both the PU.1 gene and Thy1.1 reporter. The normal PU.1 reading frame terminates at the junction of the fragment of exon 4 and the IRES, and this construct would not be expected to encode a functional PU.1 protein. Also shown are restriction enzyme sites, including a PCR-generated EcoRI site in front of the IRES, Thy1.1 cDNA, and exon 5. This extra EcoRI site allows detection of the transgene from the endogenous PU.1 gene using a 544-bp BamHI Thy1.1 cDNA probe on Southern blots to establish transgene copy number.31 (B) Map of DNase I HSs and genomic probes. The top panel is a kilobase scale. The middle panel is a map of the murine PU.1 genomic sequence in this region, showing restriction enzymes used for DNase I mapping. Also shown are the 5 coding exons (E1-E5). DNase I HSs identified in this paper are indicated below the genomic map in either bold arrows (for sites in 416B myeloid cells) or in thin arrows (for sites in BW5147 T cells); sites identified in the myeloid cells but not T cells are indicated with a minus (−) symbol. The relative positions of each of the 6 probes (P1 to P6) and the restriction enzymes used with each probe are indicated at the bottom of the figure.

Isolation and characterization of a murine PU.1 91-kb P1 genomic clone.

(A) Map of the genomic clone and strategy for insertion of the Thy1.1 reporter by homologous recombination. The top cartoon is a kilobase scale. The middle panel is a map of the murine PU.1 91-kb genomic insert (thick black line), with sequences from the P1 vector shown by the thinner black line. Shown are the relative locations of the 5 coding exons, labeled E1 to E5; restriction sites of enzymes used to map the clone; and the location of the murine equivalent of the 3′ end of the human tbp-1 gene at the extreme 5′ end of the insert. The bottom panel shows the strategy used for insertion of the Thy1.1 reporter by homologous recombination. “Genomic sequence” represents an expanded view of the murine PU.1 genomic region between exons 3 to 5. The panel below depicts the shuttle vector in which a 928-bp homologous fragment, including parts of exon 3, intron 3, and exon 4, were placed upstream of an IRES followed by the 544-bp murine Thy1.1 cDNA, a polyadenylation signal, and a 375-bp homologous portion of exon 5. “Homologous recombination” represents the final structure of the modified P1 vector, referred to as mPU.1-Thy1.1-P1 in the text. Following homologous recombination into the same region as was done for PU.1 knockout animals, we predict a genomic structure of the P1 as shown in the bottom figure in which a single transcript would encode both the PU.1 gene and Thy1.1 reporter. The normal PU.1 reading frame terminates at the junction of the fragment of exon 4 and the IRES, and this construct would not be expected to encode a functional PU.1 protein. Also shown are restriction enzyme sites, including a PCR-generated EcoRI site in front of the IRES, Thy1.1 cDNA, and exon 5. This extra EcoRI site allows detection of the transgene from the endogenous PU.1 gene using a 544-bp BamHI Thy1.1 cDNA probe on Southern blots to establish transgene copy number.31 (B) Map of DNase I HSs and genomic probes. The top panel is a kilobase scale. The middle panel is a map of the murine PU.1 genomic sequence in this region, showing restriction enzymes used for DNase I mapping. Also shown are the 5 coding exons (E1-E5). DNase I HSs identified in this paper are indicated below the genomic map in either bold arrows (for sites in 416B myeloid cells) or in thin arrows (for sites in BW5147 T cells); sites identified in the myeloid cells but not T cells are indicated with a minus (−) symbol. The relative positions of each of the 6 probes (P1 to P6) and the restriction enzymes used with each probe are indicated at the bottom of the figure.

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