Identification of the XPG Region That Causes the Onset of Cockayne Syndrome by Using Xpg Mutant Mice Generated by the cDNA-Mediated Knock-In Method
Identification of the XPG Region That Causes the Onset of Cockayne Syndrome by Using Xpg Mutant Mice Generated by the cDNA-Mediated Knock-In Method
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DOI：
10.1128/mcb.24.9.3712-3719.2004
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发表时间：
2004-05
影响因子：
5.3
通讯作者：
N. Shiomi;S. Kito;Masaki Oyama;T. Matsunaga;Y. Harada;M. Ikawa;M. Okabe;T. Shiomi
中科院分区：
文献类型：
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作者：
N. Shiomi;S. Kito;Masaki Oyama;T. Matsunaga;Y. Harada;M. Ikawa;M. Okabe;T. Shiomi
摘要 除了色素性干皮病 (XP) 之外，人类 XPG 基因突变也会导致某些患者 (XPG/CS) 早发科凯恩综合征 (CS)。此类患者中引起 CS 的突变都会产生截短的 XPG 蛋白。为了检验 XPG/CS 患者的 CS 表型（具有生长迟缓和寿命短等特征）是由 C 端截短引起的假设，我们在小鼠 XPG (Xpg) 中构建了 C 端截短的突变体（从残基 D811 到终止密码子 [XpgD811stop] 和外显子 15 [XpgΔex15] 的删除）。在XpgD811stop和XpgΔex15突变中，蛋白质的最后360个和183个氨基酸分别被删除。为了产生 Xpg 突变小鼠，我们设计了快捷敲入方法，用突变的 cDNA 片段替换基因组 DNA（cDNA 介导的敲入）。 Xpg基因组DNA片段的一半被正常的Xpg cDNA片段替换的对照小鼠具有正常的生长速度、正常的寿命、对紫外线的敏感性正常以及正常的DNA修复能力，表明部分被正常的cDNA片段替换的Xpg基因保留了正常的功能。 XpgD811stop纯合小鼠表现出生长迟缓和短寿命，但XpgΔex15纯合小鼠则没有，这表明最后360个氨基酸的缺失会导致CS表型，而最后183个氨基酸的缺失则不会。然而，XpgD811stop纯合小鼠表现出比Xpg无效突变小鼠稍微温和的CS表型，表明XpgD811stop蛋白仍然保留一些影响CS表型严重程度的Xpg功能。
ABSTRACT In addition to xeroderma pigmentosum (XP), mutations in the human XPG gene cause early onset of Cockayne syndrome (CS) in some patients (XPG/CS). The CS-causing mutations in such patients all produce truncated XPG proteins. To test the hypothesis that the CS phenotype, with characteristics such as growth retardation and a short life span in XPG/CS patients, results from C-terminal truncations, we constructed mutants with C-terminal truncations in mouse XPG (Xpg) (from residue D811 to the stop codon [XpgD811stop] and deletion of exon 15 [XpgΔex15]). In the XpgD811stop and XpgΔex15 mutations, the last 360 and 183 amino acids of the protein were deleted, respectively. To generate Xpg mutant mice, we devised the shortcut knock-in method by replacing genomic DNA with a mutated cDNA fragment (cDNA-mediated knock in). The control mice, in which one-half of Xpg genomic DNA fragment was replaced with a normal Xpg cDNA fragment, had a normal growth rate, a normal life span, normal sensitivity to UV light, and normal DNA repair ability, indicating that the Xpg gene partially replaced with the normal cDNA fragment retained normal functions. The XpgD811stop homozygous mice exhibited growth retardation and a short life span, but the XpgΔex15 homozygous mice did not, indicating that deletion of the last 360 amino acids results in the CS phenotype but deletion of the last 183 amino acids does not. The XpgD811stop homozygous mice, however, exhibited a slightly milder CS phenotype than did the Xpg null mutant mice, indicating that the XpgD811stop protein still retains some Xpg function that affects the severity of the CS phenotype.