Characterization of mitochondrial ferritin-deficient mice.
Characterization of mitochondrial ferritin-deficient mice.
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DOI：
10.1002/ajh.21872
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发表时间：
2010-12
影响因子：
12.8
通讯作者：
Fleming, Mark D.
中科院分区：
文献类型：
--
作者：
Bartnikas, Thomas B.;Campagna, Dean R.;Antiochos, Brendan;Mulhern, Howard;Pondarre, Corinne;Fleming, Mark D.
铁蛋白是高度保守的多亚基蛋白质，可以解毒并可逆地储存细胞内的铁(1)。缺乏胞质铁蛋白重链(H)的小鼠表现出胚胎致死性；条件性铁蛋白H缺失的小鼠表现出严重的组织损伤(2，3)。线粒体铁蛋白(Ftmt)在结构和功能上与铁蛋白H链(4，5)同源。铁粒母细胞性贫血患者红细胞中Ftmt的表达升高表明Ftmt在这些贫血中发挥了作用，这是一组以线粒体铁沉积为特征的遗传性和获得性疾病的异质性组[6]。虽然胞质铁蛋白H和轻链(L)在哺乳动物中普遍存在，但Ftmt mRNA主要在睾丸中发现(7，8)。Ftmt基因中缺乏铁反应元件，但存在于铁蛋白H和L的mRNAs中，提示Ftmt可能独立于铁代谢发挥作用。到目前为止，研究的重点是Ftmt过表达对组织培养、酵母和果蝇中铁超载和氧化应激的保护作用(9-15)。为了研究Ftmt在体内的作用，我们在小鼠中缺失了Ftmt基因，并检测了基线血液学、铁代谢和男性生育表型。此外，我们对他们进行维生素B6(吡哆醇)剥夺，以诱导铁粒母细胞/铁粒细胞的形成。在所有情况下，我们在缺乏线粒体铁蛋白的小鼠中没有观察到明显的缺陷。为了构建Ftmt缺陷小鼠，我们用新霉素耐药盒替换了无内含子Ftmt基因的整个开放阅读框架(ORF)(图1A)。使用靶向构建物包围的区域两侧的探针的Southern杂交表明，胚胎干细胞(ES)中的Ftmt基因座成功重组(数据未显示)。我们使用针对侧翼区和新霉素盒的聚合酶链式反应和引物确认了Ftmt的缺失(图1B)。为了检测Ftmt的表达，我们使用了逆转录聚合酶链式反应和针对β-肌动蛋白和Ftmt的特异性引物；进行了没有反转录的对照反应，以确保聚合酶产物不代表基因组DNA的扩增。由于Ftmt在睾丸中表达最丰富，我们测量了从野生型和纯合突变动物的睾丸中分离的RNA中的表达；在纯合突变动物中没有检测到表达(图1C)。我们没有检测到Ftmt在骨髓中的表达，即使在吡哆醇剥夺后也是如此(数据未显示；见下文)。
Ferritins are highly conserved multi-subunit proteins that detoxify and reversibly store intracellular iron (1). Mice lacking the cytosolic ferritin heavy chain (H) exhibit embryonic lethality; mice with a conditional ferritin H deletion exhibit severe tissue damage (2, 3). Mitochondrial ferritin (Ftmt) is structurally and functionally homologous to ferritin H chain (4, 5). Elevated Ftmt expression in erythroblasts from sideroblastic anemia patients suggests a role for Ftmt in these anemias, a heterogeneous group of inherited and acquired disorders characterized by mitochondrial iron deposition (6). While cytosolic ferritin H and light (L) chains are ubiquitous in mammals, Ftmt mRNA is found largely in the testes (7, 8). The lack of an iron responsive element in the Ftmt mRNA, while present in ferritin H and L mRNAs, suggests that Ftmt may function independently of iron metabolism. Studies to date have focused on protective effects of Ftmt overexpression against iron overload and oxidative stress in tissue culture, yeast and flies (9–15). To investigate the in vivo role of Ftmt, we deleted the Ftmt gene in mice and examined baseline hematology, iron metabolism and male fertility phenotypes. In addition, we subjected them to vitamin B6 (pyridoxine) deprivation to induce sideroblast/siderocyte formation. In all cases, we observed no significant defects in mice lacking mitochondrial ferritin.To construct Ftmt-deficient mice, we replaced the entire open reading frame (ORF) of the intronless Ftmt gene with a neomycin-resistance cassette (Fig. 1A). Southern blotting using probes flanking the region encompassed by the targeting construct indicated successful recombination at the Ftmt locus in embryonic stem (ES) cells (data not shown). We confirmed Ftmt deletion using PCR and primers specific to the flanking regions and the neomycin cassette (Fig. 1B). To measure Ftmt expression, we used reverse transcriptasepolymerase chain reaction (RT-PCR) and primers specific to β-actin and Ftmt; control reactions without reverse transcription were performed to ensure that PCR products did not represent amplification of genomic DNA. As Ftmt is most abundantly expressed in the testis, we measured expression in RNA isolated from testes of wild-type and homozygous littermates; no expression was detected in homozygous mutant animals (Fig. 1C). We could not detect Ftmt expression in bone marrow from either genotype, even after pyridoxine deprivation (data not shown; see below).
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影响因子：
4.8
作者：
Ferreira, C;Bucchini, D;Beaumont, C
通讯作者：
Beaumont, C
DOI：
10.1073/pnas.0601471103
发表时间：
2006-04-11
影响因子：
11.1
作者：
Missirlis, F;Holmberg, S;Law, JH
通讯作者：
Law, JH
DOI：
10.1016/j.biocel.2003.10.020
发表时间：
2004-10-01
影响因子：
4
作者：
Levi, S;Arosio, P
通讯作者：
Arosio, P
影响因子：
3.5
作者：
Campanella, A;Isaya, G;Levi, S
通讯作者：
Levi, S
影响因子：
3.3
作者：
Tangjarukij, Chanthana;Navasumrit, Panida;Ruchirawat, Mathuros
通讯作者：
Ruchirawat, Mathuros