ANALYSIS OF TRANSCRIPTION AND ESTROGEN INSENSITIVITY IN THE FEMALE MOUSE AFTER TARGETED DISRUPTION OF THE ESTROGEN-RECEPTOR GENE
ANALYSIS OF TRANSCRIPTION AND ESTROGEN INSENSITIVITY IN THE FEMALE MOUSE AFTER TARGETED DISRUPTION OF THE ESTROGEN-RECEPTOR GENE
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DOI：
10.1210/me.9.11.1441
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发表时间：
1995-11-01
影响因子：
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通讯作者：
KORACH, KS
中科院分区：
文献类型：
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作者：
COUSE, JF;CURTIS, SW;KORACH, KS
我们在小鼠胚胎干细胞中采用同源重组来破坏雌激素受体（ER）基因。随后产生的小鼠是纯合子的基因破坏，称为ERKO，拥有没有明显的野生型ER通过蛋白质印迹分析。然而，残留的高亲和力结合的存在下，从ERKO女性子宫提取物中检测到的[H-3]雌二醇结合试验和蔗糖梯度，促使进一步调查的转录和翻译产物从破坏的ER基因。通过逆转录-聚合酶链反应分析ERKO子宫信使RNA（mRNA）表明，虽然没有全长野生型ER mRNA的存在，两个较小的成绩单，标记E1和E2，被确定和部分测序。两种ERKO转录物都是剪接变体，其导致破坏性NEO序列从mRNA中部分或完全去除。在ERKO-ES变体中，这导致移码和下游至少两个终止密码子的产生。在ERKO-E1变异体中，ER阅读框架被保留，并编码一个较小的突变ER，这可能是残留雌二醇结合的来源。当这种突变形式在体外过表达和表征时，它导致预测大小的较小蛋白质，与野生型ER相比，其具有显著降低的雌激素依赖性转录活性。尽管有残留量的受损ER变体，雌激素治疗未能诱导已知的雌激素作用的子宫标志物，如DNA合成增加，孕酮受体，乳铁蛋白和葡萄糖-6-磷酸脱氢酶基因的转录，证实了女性ERKO的雌激素不敏感性。此外，ERKO女性的血清雌二醇水平比野生型高10倍以上，与激素不敏感综合征一致。
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 [H-3]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-ES 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.