Gene manipulation of heme synthetic pathway enzymes

血红素生物合成途径包含一系列组织特异性酶，包括ALAS-E（Alas 2）。我们的目的是通过建立ALAS-E基因敲除小鼠系，阐明血红素合成缺陷引起的先天性疾病，如X连锁铁粒幼细胞性贫血（XLSA）的发病机制。通过基因打靶实验建立ALAS-E基因敲除小鼠品系。ALAS-E缺陷小鼠具有胚胎致死性。致死的原因似乎是有缺陷的原始造血。ALAS-E-null ES细胞的嵌合分析揭示了与在人XLSA患者中观察到的那些相似的铁粒幼细胞的存在。因此，认为环状铁粒幼细胞可能来源于ALAS-E基因敲除的ES细胞。然后，我们产生了转基因（TG）小鼠系表达ALAS-E突变体分子分离的XLSA患者的人样本下的GATA 1-造血调控结构域（G1 HRD）的调控。当G1 HRD-ALAS-Emut TG小鼠与ALAS-E-null背景杂交时，ALAS-E-null小鼠的严重表型仅得到部分挽救，表明需要ALAS-E的完全活性来完全挽救敲除表型。结果表明ALAS-E突变分子功能受损。这些结果表明血红素合成与红细胞生成密切相关。

Heme biosynthesis pathway contains a battery of tissue-specific enzymes, including ALAS-E (Alas2). Our purpose is to clarify the pathogenesis of congenital diseases caused by the defective heme synthesis, such as X-linked sideroblastic anemia (XLSA), through generating an ALAS-E knockout line of mice. We generated ALAS-E knockout mouse line through gene targeting experiments. The ALAS-E-deficient mice were embryonic lethal. The cause of the lethality appeared to be the defective primitive hematopoiesis. Chimeric analysis of ALAS-E-null ES cells revealed the presence of sideroblasts that are similar to those observed in human XLSA patients. It was highly expected that the ring sideroblasts were originated from the ALAS-E-null ES cells. We then generated transgenic (TG) mouse lines expressing ALAS-E mutant molecule isolated from the human samples of XLSA patients under the regulation of GATA1-hematopoietic regulatory domain (G1HRD). When G1HRD-ALAS-Emut TG mice were crossed into ALAS-E-null background, severe phenotypes of ALAS-E-null mice were rescued only partially, suggesting that full-activity of ALAS-E is required to fully rescue the knockout phenotype. The results indicate that the ALAS-E mutant molecule is functionally impaired. These results thus demonstrate that close relationship between heme synthesis and erythropoiesis.