Mouse estrogen receptor gene knock out: Molecular characterization and resulting phenotypes

Abstract

The estrogen receptor (ER) is thought to play a crucial role in the regulation of many l i f e processes, including development, reproduction and normal physiology. Because there have been no known mutations of the estrogen receptor in normal t issue of humans or animals, i t s presence and t issue d is t r ibu t ion is thought to be essential for surv iva l . Using the techniques of homologous recombination, we have disrupted the ER gene in mouse embryonic stem cel ls and have produced a l ine of transgenic mice possessing the altered ER gene. The mouse ER gene was disrupted by insert ing a 1.8kb PGK-Neomycin sequence exon 2, approximately 280bp downstream of the s tar t codon. The correct target ing of the disruption was demonstrated by both Southern blot analysis and polymerase chain reaction (PCR). Western blot analysis of uterine preparations from females that are homozygous for the ER gene disruption showed no detectable ER protein. Heterozygotes had one hal f the level of ER protein. Treatment with estradiol at 40ug/kg/day for three days resulted in a 3-4 fold increase in uterine wet weight and vaginal corn i f icat ion in the wi ld type females but e l i c i t ed no s imi la r response in the homozygous mutant females. This data was fur ther supported by a 3H-Thymidine incorporation assay on uterine t issue of s im i la r l y treated mice. Lactoferr in, an estrogen responsive gene in the uterus, was also assayed by northern blot . Ovariectomized wi ld type mice treated with a single estradiol in ject ion showed a 350-fold induction in lac to fer r in mRNA, while ovariectomized homozygous ER mutant females showed no detectable response. Both male and female animals survive to adulthood with normal gross external phenotypes. As expected, females are i n f e r t i l e and demonstrate hypoplastic uter i and hyperemic ovaries with no apparent corpora lutea. Males are also i n f e r t i l e , with atrophy of the testes. Preliminary analysis of the mammary glands of ER mutant females at four months of age showed a pr imi t ive ductal rudiment that extended only 5-6mm into the fat pad compared to a f u l l y developed ductal tree in wild type s ib l ings. Also absent were the terminal end buds seen during normal ductal morphogenesis. These findings demonstrate that the absence of functional estrogen receptor is not le tha l . Although the reproductive capabi i t ies have been al tered, prenatal development of the reproductive t racts of both sexes appears to be independent of estrogen receptor medicated responses. This animal should provide an ef fect ive in vi~o model to discern the role of ER in a var ie ty of physiological processes.