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of E2 gene knockout mice\nTo create E2 knockout mice, we used gene targeting in mouse ES cells to disrupt the E2 gene. The overall strategy for disrupting the E2 gene is illustrated in Fig. 1A. The gene targeting construct was designed to replace a 1.67 kb EcoRV/Smal genomic DNA fragment encompassing part of Exon 4 and all of Exon 5 with the PGKneo selectable marker cassette. Of 522 ES cell clones screened for targeting by Southern blot analysis, 29 (5%) displayed the predicted restriction fragment length polymorphisms indicative of gene targeting at the E2 locus. As illustrated in Fig. 1A and 1B, an E2 Exon 6 specific probe, which is external to the targeting construct, hybridizes to only a ~16 kb BglI restriction fragment from the wild type allele in the parental R1 ES cell line. In correctly targeted ES cells, this probe also hybridizes to a ~11 kb BglI restriction fragment. Targeting was confirmed with several additional restriction enzymes and probes (data not shown).\nCorrectly targeted ES cells were microinjected into blastocysts to produce chimeric mice. Chimerics were bred to C57BL/6J mice. Following germline transmission of the targeted allele, heterozygous mice were interbred to produce wild type (+/+), heterozygous (+/-) and homozygous (-/-) animals. Mice were genotyped by Southern blot analysis. The Exon 6 specific probe hybridized to only a ~16 kb BglI restriction fragment in +/+ mice, a ~11 kb BglI fragment in -/- mice, and to both of these fragments in +/- mice (Fig. 1B).\nMice homozygous for the E2 mutation were born at the expected frequency. Genotype analysis of pups derived from +/- by +/- matings revealed that +/+, +/-, and -/- mice were present at nearly the expected 1:2:1 frequency. Of the initial 60 animals genotyped, 19 (32%) were +/+, 27 (45%) were +/-, and 14 (23%) were -/-. Thus, the E2 gene was dispensable for normal embryonic development. However, as expected, nearly all homozygous mice died in the perinatal period. Immediately following birth, homozygous pups were indistinguishable from their +/+ and +/- littermates; they were vigorous, active and able to suckle. By mid to late day on postnatal day one, most -/- pups became moribund and were readily identifiable as they were lethargic, pale, and exhibited gasping respiratory movements. With few exceptions, -/- pups died within 72 hours of birth. We have observed one rare -/- pup that survived to postnatal day 13. The reason for the prolonged survival of this pup is unknown."}

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of E2 gene knockout mice\nTo create E2 knockout mice, we used gene targeting in mouse ES cells to disrupt the E2 gene. The overall strategy for disrupting the E2 gene is illustrated in Fig. 1A. The gene targeting construct was designed to replace a 1.67 kb EcoRV/Smal genomic DNA fragment encompassing part of Exon 4 and all of Exon 5 with the PGKneo selectable marker cassette. Of 522 ES cell clones screened for targeting by Southern blot analysis, 29 (5%) displayed the predicted restriction fragment length polymorphisms indicative of gene targeting at the E2 locus. As illustrated in Fig. 1A and 1B, an E2 Exon 6 specific probe, which is external to the targeting construct, hybridizes to only a ~16 kb BglI restriction fragment from the wild type allele in the parental R1 ES cell line. In correctly targeted ES cells, this probe also hybridizes to a ~11 kb BglI restriction fragment. Targeting was confirmed with several additional restriction enzymes and probes (data not shown).\nCorrectly targeted ES cells were microinjected into blastocysts to produce chimeric mice. Chimerics were bred to C57BL/6J mice. Following germline transmission of the targeted allele, heterozygous mice were interbred to produce wild type (+/+), heterozygous (+/-) and homozygous (-/-) animals. Mice were genotyped by Southern blot analysis. The Exon 6 specific probe hybridized to only a ~16 kb BglI restriction fragment in +/+ mice, a ~11 kb BglI fragment in -/- mice, and to both of these fragments in +/- mice (Fig. 1B).\nMice homozygous for the E2 mutation were born at the expected frequency. Genotype analysis of pups derived from +/- by +/- matings revealed that +/+, +/-, and -/- mice were present at nearly the expected 1:2:1 frequency. Of the initial 60 animals genotyped, 19 (32%) were +/+, 27 (45%) were +/-, and 14 (23%) were -/-. Thus, the E2 gene was dispensable for normal embryonic development. However, as expected, nearly all homozygous mice died in the perinatal period. Immediately following birth, homozygous pups were indistinguishable from their +/+ and +/- littermates; they were vigorous, active and able to suckle. By mid to late day on postnatal day one, most -/- pups became moribund and were readily identifiable as they were lethargic, pale, and exhibited gasping respiratory movements. With few exceptions, -/- pups died within 72 hours of birth. We have observed one rare -/- pup that survived to postnatal day 13. The reason for the prolonged survival of this pup is unknown."}

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The overall strategy for disrupting the E2 gene is illustrated in Fig. 1A. The gene targeting construct was designed to replace a 1.67 kb EcoRV/Smal genomic DNA fragment encompassing part of Exon 4 and all of Exon 5 with the PGKneo selectable marker cassette. Of 522 ES cell clones screened for targeting by Southern blot analysis, 29 (5%) displayed the predicted restriction fragment length polymorphisms indicative of gene targeting at the E2 locus. As illustrated in Fig. 1A and 1B, an E2 Exon 6 specific probe, which is external to the targeting construct, hybridizes to only a ~16 kb BglI restriction fragment from the wild type allele in the parental R1 ES cell line. In correctly targeted ES cells, this probe also hybridizes to a ~11 kb BglI restriction fragment. Targeting was confirmed with several additional restriction enzymes and probes (data not shown).\nCorrectly targeted ES cells were microinjected into blastocysts to produce chimeric mice. Chimerics were bred to C57BL/6J mice. Following germline transmission of the targeted allele, heterozygous mice were interbred to produce wild type (+/+), heterozygous (+/-) and homozygous (-/-) animals. Mice were genotyped by Southern blot analysis. The Exon 6 specific probe hybridized to only a ~16 kb BglI restriction fragment in +/+ mice, a ~11 kb BglI fragment in -/- mice, and to both of these fragments in +/- mice (Fig. 1B).\nMice homozygous for the E2 mutation were born at the expected frequency. Genotype analysis of pups derived from +/- by +/- matings revealed that +/+, +/-, and -/- mice were present at nearly the expected 1:2:1 frequency. Of the initial 60 animals genotyped, 19 (32%) were +/+, 27 (45%) were +/-, and 14 (23%) were -/-. Thus, the E2 gene was dispensable for normal embryonic development. However, as expected, nearly all homozygous mice died in the perinatal period. Immediately following birth, homozygous pups were indistinguishable from their +/+ and +/- littermates; they were vigorous, active and able to suckle. By mid to late day on postnatal day one, most -/- pups became moribund and were readily identifiable as they were lethargic, pale, and exhibited gasping respiratory movements. With few exceptions, -/- pups died within 72 hours of birth. We have observed one rare -/- pup that survived to postnatal day 13. The reason for the prolonged survival of this pup is unknown."}