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1 E2 gene knockout mouse production. A, Gene targeting strategy used for targeting the E2 locus in mouse ES cells. The targeting construct was designed to delete 1.67 kb of sequence between an EcoRV site in Exon 4 and a Smal site in intron 5. The wild type E2 gene contains an ~16 kb BglI restriction fragment that hybridizes to the Exon 6 specific probe. A correctly targeted E2 locus harbors an ~11 kb BglI restriction fragment that hybridizes to the same probe. Note that the probe will not detect random integration of the targeting vector because it is external to the targeting vector. B, Southern blot analysis of Bgll digested genomic DNA derived from the parental wild type ES cell line (R1), a heterozygous targeted ES cell line (362), and from wild type (+/+), heterozygous (+/-) and homozygous knockout (-/-) mice. The blot was hybridized with an Exon 6 specific probe. C, Immunohistochemical analysis of fresh frozen liver sections from control (+/+) and E2 knockout (-/-) postnatal day 1 mouse pups. Sections were stained for E2 using an E2 specific antibody (green) and a nuclear stain (blue). Note the complete absence of E2 immunoreactivity in the section from the knockout mouse. D, Similar results were observed upon immunohistochemical analysis of primary mouse embryonic fibroblasts (MEFs). Note that the readily detectable signal for E2 in the control cells was present in a pattern characteristic of mitochondria, the subcellular location of BCKDH."}

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1 E2 gene knockout mouse production. A, Gene targeting strategy used for targeting the E2 locus in mouse ES cells. The targeting construct was designed to delete 1.67 kb of sequence between an EcoRV site in Exon 4 and a Smal site in intron 5. The wild type E2 gene contains an ~16 kb BglI restriction fragment that hybridizes to the Exon 6 specific probe. A correctly targeted E2 locus harbors an ~11 kb BglI restriction fragment that hybridizes to the same probe. Note that the probe will not detect random integration of the targeting vector because it is external to the targeting vector. B, Southern blot analysis of Bgll digested genomic DNA derived from the parental wild type ES cell line (R1), a heterozygous targeted ES cell line (362), and from wild type (+/+), heterozygous (+/-) and homozygous knockout (-/-) mice. The blot was hybridized with an Exon 6 specific probe. C, Immunohistochemical analysis of fresh frozen liver sections from control (+/+) and E2 knockout (-/-) postnatal day 1 mouse pups. Sections were stained for E2 using an E2 specific antibody (green) and a nuclear stain (blue). Note the complete absence of E2 immunoreactivity in the section from the knockout mouse. D, Similar results were observed upon immunohistochemical analysis of primary mouse embryonic fibroblasts (MEFs). Note that the readily detectable signal for E2 in the control cells was present in a pattern characteristic of mitochondria, the subcellular location of BCKDH."}

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