Molecular and biochemical characterization of Mottled-dappled, an embryonic lethal Menkes disease mouse model.
Molecular and biochemical characterization of Mottled-dappled, an embryonic lethal Menkes disease mouse model.
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DOI：
10.1016/j.ymgme.2014.10.001
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发表时间：
2014-12
影响因子：
3.8
通讯作者：
Kaler, Stephen G.
中科院分区：
文献类型：
--
作者：
Haddad, Marie Reine;Patel, Keyur D.;Sullivan, Patricia H.;Goldstein, David S.;Murphy, Kevin M.;Centeno, Jose A.;Kaler, Stephen G.
斑点-斑点（Mo-dp）是门克斯病的小鼠模型，其由ATP 7A的小鼠直系同源物Atp 7a的5'区域中的大的先前未表征的缺失引起。受影响的突变体在胚胎第17天在子宫内死亡，并显示肋骨弯曲和增厚，胸部和骨盆带和四肢扭曲。为了表征该等位基因，我们在小鼠X染色体上设计了一个定制的4× 180 K微阵列，并使用从正常和携带Mo-dp女性中提取的DNA进行比较基因组杂交，并确定了一个约9 kb的缺失。我们使用PCR对断裂点进行精细定位，并扩增出630 bp的连接片段。连接片段的测序揭示了确切的断裂点位置，并且Mo-dp缺失精确地为8，990 bp，包括Atp 7a的启动子区域中的约2kb。Mo-dp杂合子脑的蛋白质印迹分析显示Atp 7a蛋白的量减少，这与由于一个等位基因上的启动子区域缺失而导致的表达减少一致。在杂合子女性，脑铜水平往往低于野生型相比，而神经化学分析显示较高的二羟基苯乙酸：二羟基苯乙二醇（DOPAC：DHPG）和多巴胺：去甲肾上腺素（DA：NE）比正常（p=0.002和0.029，分别），符合部分缺乏多巴胺-β-羟化酶，铜依赖性酶。与野生型雌性相比，杂合雌性的体重没有显着差异。我们的研究结果描绘了Mo-dp突变的分子细节，并首次定义了该等位基因杂合子女性携带者的新生化发现。
Mottled-dappled (Mo-dp) is a mouse model of Menkes disease caused by a large, previously uncharacterized deletion in the 5' region of Atp7a, the mouse ortholog of ATP7A. Affected mutants die in utero at embryonic day 17, and show bending and thickening of the ribs and distortion of the pectoral and pelvic girdles and limbs. To characterize this allele, we designed a custom 4×180K microarray on the mouse X chromosome and performed comparative genomic hybridization using extracted DNA from normal and carrier Mo-dp females, and identified an approximately 9 kb deletion. We used PCR to fine-map the breakpoints and amplify a junction fragment of 630 bp. Sequencing of the junction fragment disclosed the exact breakpoint locations and that the Mo-dp deletion is precisely 8,990 bp, including approximately 2 kb in the promoter region of Atp7a. Western blot analysis of Mo-dp heterozygotes brains showed diminished amounts of Atp7a protein, consistent with reduced expression due to the promoter region deletion on one allele. In heterozygous females, brain copper levels tended to be lower compared to wild type whereas neurochemical analyses revealed higher dihydroxyphenylacetic acid: dihydroxyphenylglycol (DOPAC: DHPG) and dopamine: norepinephrine (DA:NE) ratios compared to normal (p=0.002 and 0.029, respectively), consistent with partial deficiency of dopamine-beta-hydroxylase, a copper-dependent enzyme. Heterozygous females showed no significant differences in body weight compared to wild type females. Our results delineate the molecular details of the Mo-dp mutation for the first time and define novel biochemical findings in heterozygous female carriers of this allele.
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影响因子：
3.8
作者：
Liu, PC;Chen, YW;Kaler, SG
通讯作者：
Kaler, SG
影响因子：
3.7
作者：
Wang Y;Zhu S;Weisman GA;Gitlin JD;Petris MJ
通讯作者：
Petris MJ
影响因子：
38.1
作者：
Kaler, Stephen G.
通讯作者：
Kaler, Stephen G.
影响因子：
2.7
作者：
Czyzyk, TA;Ning, Y;Pintar, JE
通讯作者：
Pintar, JE
影响因子：
64.8
作者：
THOMAS, SA;MATSUMOTO, AM;PALMITER, RD
通讯作者：
PALMITER, RD