Proc. Natl. Acad. Sci. USA Vol. 90, pp. 11162-11166, December 1993 Genetics Alteration of reproductive function but not prenatal sexual development after insertional disruption of the mouse estrogen receptor gene (homologous recombination/gene targeting/fertility) DENNIS B. LUBAHN*tt§, JEFFREY S. MOYER*, THOMAS S. GOLDING1II, JOHN F. COUSE¶, KENNETH S. KoRACH1**, AND OLIVER SMITHIES* Departments of *Pathology and tPediatrics and tLaboratories for Reproductive Biology, University of North Carolina, Chapel Hill, NC 27599; and $Receptor Biology Section and I"Gamete Biology Section, Laboratory of Reproduction and Developmental Toxicology, National Institute of Environmental Health Sciences, National Institutes of Health, P.O. Box 12233, Research Triangle Park, NC 27709 Contributed by 0. Smithies, September 1, 1993 ABSTRACT Estrogen receptor and its ligand, estradiol, Mutations in genes for the androgen receptor protein or for have long been thought to be essential for survival, fertility, and androgen synthetic enzymes result in abnormalities in male female sexual differentiation and development. Consistent with sexual differentiation and development (5-7) with little effect this proposed crucial role, no human estrogen receptor gene in females except for a decrease in fertility (8). mutations are known, unlike the androgen receptor, where The role ofestrogen action in prenatal sexual development, many loss of function mutations have been found. We have however, is controversial. Endocrinological evidence for the generated mutant mice lacking responsiveness to estradiol by importance ofestrogens in sexual development is found in rat disrupting the estrogen receptor gene by gene targeting. Both and rabbit embryogenesis studies (9, 10), where estrogen male and female animals survive to adulthood with normal synthesis is activated in male and female embryos at the time gross external phenotypes. Females are infertile; males have a ofblastocyst implantation in the uterus. Recently, ER mRNA decreased fertility. Females have hypoplastic uteri and hyper- has been detected by the very sensitive reverse transcrip- emic ovaries with no detectable corpora lutea. In adult wild- tase/PCR technique in blastocysts and two-cell-stage em- type and heterozygous females, 3-day estradiol treatment at 40 bryos (11). In contrast, Jost (12), in a series of classical organ ,pg/kg stimulates a 3- to 4-fold increase in uterine wet weight ablation experiments, demonstrated that fetal gonadectomy and alters vaginal cornification, but the uteri and vagina do not of mammalian males and females resulted in both sexes respond in the animals with the estrogen receptor gene dis- developing as phenotypic females, suggesting that estrogen is ruption. Prenatal male and female reproductive tract devel- not needed for female sexual development. However, the opment can therefore occur in the absence ofestradiol receptor- presence ofmaternal and placental estrogens left the question mediated responsiveness. of estrogen's importance in prenatal sexual development unresolved. The estrogen receptor (ER) is a member of the steroid Nevertheless, there is no doubt that estrogen plays a receptor superfamily ofligand-activated transcription factors central role in normal postnatal female physiology and in (1). ER and its hormone ligand, the female sex steroid female pathology, where its importance in breast and uterine 17,B-estradiol, have long been known to play critical roles in cancer, osteoporosis, and cardiovascular disease is well the development of feminine secondary sexual characteris- known although poorly understood. Besides the normal tics as well as in the female reproductive cycle, infertility, and physiology and endocrinology of estrogen action, the mech- maintenance of pregnancy. Estradiol is also thought to be anisms for hormonal stimulation have also been postulated to essential for embryonic and fetal development (2). involve other cellular signaling mechanisms (13-15). Supporting evidence for the crucial role of estrogen is Because of the various uncertainties regarding the roles of based on pharmacological and genetic data that estrogen estrogen, we decided to disrupt the ER gene in mouse antagonists and inhibitors ofestrogen synthesis interfere with embryonic stem cells by homologous recombination, so as to placental function and cause abortions (2). Overexpression create an animal lacking a functional estrogen receptor. mutations in the mammalian estrogen biosynthetic enzyme, Experimental development of this animal model was ex- aromatase, have been reported (3) to lead to gynecomastia in pected to provide a clear role for ER action in a variety of males because of excess conversion of androgens to estro- systems under physiological conditions. gens. Lack of function mutations in human placental aro- matase have been reported to cause maternal virilization and MATERIALS AND METHODS fetal pseudohermaphroditism in female offspring (4). This observation suggested the need for estrogens in normal Gene-Targeting Plasmid. The mouse ER gene has been prenatal sexual development. This interpretation may be cloned and found to have nine exons (16). Utilizing the confounded, however, by the effects ofsecondarily increased published sequence and PCR (17) with oligonucleotide primer androgen concentrations. In addition, the absence of re- pairs 1-2 and 3-4 (see below), we amplified two DNA ported human ER mutations supports the suggestion that if fragments from the most N-terminal exon of the mouse ER natural ER functional mutations do occur, they may be lethal gene. The two amplified fragments were used as probes to in eutherian animals (2). allow the cloning of a 10-kb BamHI fragment containing the Lack of known ER mutations is contrasted with the situation in regards to the male sex steroids, the androgens. Abbreviations: ER, estrogen receptor; TK, thymidine kinase; PGK, phosphoglycerate kinase; ES, embryonic stem; Neo, neomycin resistance. The publication costs of this article were defrayed in part by page charge §Present address: Department of Cellular Biochemistry, Glaxo Inc. payment. This article must therefore be hereby marked "advertisement" Research Institute, Durham, NC 27709. in accordance with 18 U.S.C. §1734 solely to indicate this fact. **To whom reprint requests should be addressed. 11162 Downloaded by guest on September 25, 2021 Genetics: Lubahn et al. Proc. Natl. Acad. Sci. USA 90 (1993) 11163 second exon of the mouse ER gene from a lambda Dash pared from these plates was pooled into groups of four and (Stratagene) library constructed from BamHI-digested DNA was screened by PCR amplification to detect targeting (22). obtained from E14TG2a cells (18). To assemble the targeting PCR Primers. Oligo 1, used for making the 5' probe during a gene driven by the phosphoglyc- cloning of the ER gene: 5'-CGCTGCTGAGCCCTCT- construct (Fig. 1B), Neo during erate kinase (PGK) promoter and having a PGK poly(A) GCGTG-3'. Oligo 2, used for making the 5' probe was cloned in a 5'-to-3' orientation in a cloning of ER gene: 5'-GTTGAACTCGTAGGCGGCGC- addition signal (19) Oligo 3, used for making the 3' probe during Not I site within exon 2 contained in a 7.5-kb BamHI/Spe I CCTC-3'. This inser- cloning of the ER gene and for determination of the presence fragment, made from the 10-kb BamHI fragment. of the wild-type ER gene: 5'-CGGTCTACGGCCAG- tion disrupts the reading frame. A flanking herpes simplex TCGGGCACC-3'. Oligo 4, used for making the 3' probe TK gene driven by the same promoter and enhancer was during cloning of the ER gene and for determination of the added to the 3' end of the BamHI/Spe I fragment (20). presence of the wild-type ER gene: 5'-GTAGAAGGCGG- Because of difficulty in cloning of the 5' 7-kb BamHI/Not I GAGGGCCGGTGTC-3'. Oligo 5, from sequence in the 3' fragment in plasmids, the targeting construct was assembled end of the PGK gene, used for determination of Neo disrup- in a lambda Dash bacteriophage vector. The Neo gene/Not tion of the ER gene after recombination: 5'-TTCCACATA- I/Spe I fragment and TK gene were first cloned in pBlue- CACTTCATTCTCA-3'. Oligo 6, from sequence external to script (Stratagene) and then subcloned in a lambda Dash the targeting construct in intron 2 of the ER gene, used for vector containing the ER 7-kb BamHI/Not I fragment. A determination of Neo disruption of the ER gene after recom- BamHI site in the Neo gene was destroyed prior to cloning bination: 5'-CTCCACTGGCCTCAAACACCTG-3'. in lambda Dash vector so that BamHI could be used to PCR Amplification. The 3' end ofthe PGK gene, containing remove the A arms. the poly(A) addition signal, was sequenced. From this se- Embryonic Stem (ES) Cells. The ES cell line E14TG2a, quence, oligo 5 was designed for use in conjunction with oligo derived originally from a 129/J strain mouse (18), was cul- 6 to amplify a 649-bp fragment diagnostic of a successfully tured on primary embryonic fibroblast feeder layers previ- targeted ER gene (see Fig. 1). For PCR screening the DNA ously irradiated with 3000 rads (30 Gy); the fibroblasts were was from pools of four colonies of ES cells. PCR was also isolated from neomycin-resistant embryos (21) and were used to distinguish normal, heterozygous, and homozygous mutant animals (see Fig. 2). resistant to G418 (Sigma). ES cells were injected into Electroporation. Electroporation of about 1 x 107 ES cells Mouse Breeding. The targeted medium blastocysts from C57BL/6J mothers and were returned to was in 0.5 ml of Dulbecco's modified Eagle's pseudopregnant C57BL/6J hosts to complete their develop- (DMEM)/15% fetal bovine serum/0.1 mM 2-mercaptoetha- ment. Chimeras were identified by coat color and males were nol/2 mM glutamine with 5 nM targeting construct with 1-sec bred to C57BL/6J females. Tail DNA from agouti coat color discharge from a 150- to 250-,uF capacitor charged to 250-400 F1 offspring was screened by PCR with primers 5 and 6 for V. In early electroporations the A arms were removed from presence of the targeted ER gene (a 649-bp fragment).