title: Analysis of transcription and estrogen insensitivity in the female mouse after targeted disruption of the estrogen receptor gene.
abstract: We employed homologous recombination in mouse embryonic stem cells to disrupt the estrogen receptor (ER) gene. Subsequently generated mice that are homozygous for the gene disruption, termed ERKO, possess no demonstrable wild-type ER by Western blot analysis. However, the presence of residual high affinity binding, as detected by [3H]estradiol binding assays and sucrose gradients in uterine extracts from ERKO females prompted further investigation of transcription and translation products from the disrupted ER gene. Analysis of ERKO uterine messenger RNA (mRNA) by reverse transcriptase-polymerase chain reaction demonstrated that although no full-length wild-type ER mRNA was present, two smaller transcripts, labeled E1 and E2, were identified and partially sequenced. Both ERKO transcripts are splicing variants that result in the disrupting NEO sequence being partially or completely removed from the mRNA. In the ERKO-E2 variant, this results in a frame shift and the creation of at least two stop codons downstream. In the ERKO-E1 variant, the ER reading frame is preserved and encodes for a smaller mutant ER that could be the source of the residual estradiol binding. When this mutant form is overexpressed and characterized in vitro, it results in a smaller protein of the predicted size that possesses significantly reduced estrogen-dependent transcriptional activity compared with that of the wild-type ER. Despite residual amounts of an impaired ER variant, estrogen insensitivity in the female ERKOs was confirmed by the failure of estrogen treatment to induce known uterine markers of estrogen action, such as increased DNA synthesis, and transcription of the progesterone receptor, lactoferrin, and glucose-6-phosphate dehydrogenase genes. Furthermore, serum levels of estradiol in the ERKO female are more than 10-fold higher than those in the wild type, consistent with a syndrome of hormone insensitivity.
authors.affiliation: Receptor Biology Section, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
authors.forename: J F
authors.initials: JF
authors.lastname: Couse
authors.forename: S W
authors.initials: SW
authors.lastname: Curtis
authors.forename: T F
authors.initials: TF
authors.lastname: Washburn
authors.forename: J
authors.initials: J
authors.lastname: Lindzey
authors.forename: T S
authors.initials: TS
authors.lastname: Golding
authors.forename: D B
authors.initials: DB
authors.lastname: Lubahn
authors.forename: O
authors.initials: O
authors.lastname: Smithies
authors.forename: K S
authors.initials: KS
authors.lastname: Korach
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002461:Cell Line, Transformed; D002522:Chlorocebus aethiops; D004351:Drug Resistance / Q000235:genetics; D004958:Estradiol / Q000097:blood / Q000494:pharmacology*; D005260:Female; D016368:Frameshift Mutation; D005786:Gene Expression Regulation / Q000187:drug effects; D018390:Gene Targeting; D005954:Glucosephosphate Dehydrogenase / Q000096:biosynthesis / Q000235:genetics; D007781:Lactoferrin / Q000096:biosynthesis / Q000235:genetics; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D010052:Ovariectomy; D011374:Progesterone / Q000097:blood; D014176:Protein Biosynthesis / Q000187:drug effects; D012326:RNA Splicing; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D011960:Receptors, Estrogen / Q000096:biosynthesis / Q000172:deficiency / Q000235:genetics* / Q000502:physiology; D011980:Receptors, Progesterone / Q000096:biosynthesis / Q000235:genetics; D013234:Stem Cells; D014158:Transcription, Genetic / Q000187:drug effects*; D014162:Transfection; D014599:Uterus / Q000187:drug effects / Q000378:metabolism
chemical_list: D012333:RNA, Messenger; D011960:Receptors, Estrogen; D011980:Receptors, Progesterone; D011374:Progesterone; D004958:Estradiol; D005954:Glucosephosphate Dehydrogenase; D007781:Lactoferrin