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genomic DNA subclone of Strain 129/SvJ DNA that contained a portion of Exons 4 and 5 of the E2 gene and flanking DNA was obtained from a P1 phage library from Genome Systems, Inc., (St. Louis, MO; '3-Hit Mouse ES Library'). A positive/negative replacement type gene targeting vector [21] was created by replacing a 1.67 kb EcoRV-Smal fragment that included a portion of Exon 4 and all of Exon 5 with the PGKneo marker gene (See Fig. 1A) from the pPNT vector [22]. The targeting construct was linearized with NotI and electroporated into R1 ES cells [23] under conditions described previously [24]. ES cells were selected with G418 (300 μg/ml; Life Technologies Inc., Gaithersburg, MD) and gancyclovir (2 μM; gift of Syntex, Palo Alto, CA). Doubly resistant clones were screened for gene targeting by Southern blot analysis of BglI digested genomic DNA. Blots were hybridized with an Exon 6 specific probe that was external to the gene targeting construct. Correctly targeted ES cells were injected into C57BL/6J blastocysts to produce chimeric mice using standard procedures. Heterozygous offspring from germline competent chimeras were intercrossed to produce wild type (+/+), heterozygous (+/-) and homozygous knockout (-/-) mice. At all generations, +/- breeding pairs were used. Results presented here are from mice derived from the F2+ generations. All animals were of a mixed C57BL/6J × Strain 129Sv/SvJ genetic background."}

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A positive/negative replacement type gene targeting vector [21] was created by replacing a 1.67 kb EcoRV-Smal fragment that included a portion of Exon 4 and all of Exon 5 with the PGKneo marker gene (See Fig. 1A) from the pPNT vector [22]. The targeting construct was linearized with NotI and electroporated into R1 ES cells [23] under conditions described previously [24]. ES cells were selected with G418 (300 μg/ml; Life Technologies Inc., Gaithersburg, MD) and gancyclovir (2 μM; gift of Syntex, Palo Alto, CA). Doubly resistant clones were screened for gene targeting by Southern blot analysis of BglI digested genomic DNA. Blots were hybridized with an Exon 6 specific probe that was external to the gene targeting construct. Correctly targeted ES cells were injected into C57BL/6J blastocysts to produce chimeric mice using standard procedures. Heterozygous offspring from germline competent chimeras were intercrossed to produce wild type (+/+), heterozygous (+/-) and homozygous knockout (-/-) mice. At all generations, +/- breeding pairs were used. Results presented here are from mice derived from the F2+ generations. All animals were of a mixed C57BL/6J × Strain 129Sv/SvJ genetic background."}

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