Targeted chromosomal cleavage and mutagenesis in Drosophila using zinc-finger nucleases.
Targeted chromosomal cleavage and mutagenesis in Drosophila using zinc-finger nucleases.
复制标题
使用锌指核酸酶对果蝇进行靶向染色体切割和诱变。
DOI：
10.1093/genetics/161.3.1169
复制
发表时间：
2002
期刊：
影响因子：
3.3
通讯作者：
Carroll,Dana
中科院分区：
文献类型：
--
作者：
Bibikova,Marina;Golic,Mary;Golic,KentG;Carroll,Dana
锌指核酸酶(ZFN)是由Cys2His2锌指组成的DNA结合结构域和非特异性DNA裂解结构域的杂交体。由于锌指可以被操纵来识别广泛的序列，这些酶有可能直接切割到任意选择的靶标。我们通过设计一对ZFN来测试这一想法，该ZFN识别果蝇黄色(Y)基因中的一个独特位置。当这些核酸酶在发育中的幼虫中表达时，它们会导致它们基因中特定的体细胞突变。在表达两种核酸酶的男性中，大约有一半观察到了这些体细胞嵌合体。从5.7%的男性身上发现了生殖线突变，但没有一名女性被检测到。DNA序列测定表明，所有突变都是精确定位于设计靶点的小片段缺失和/或插入。这些正是靶标双链切割后非同源末端连接(NHEJ)预期的变化类型。这种方法有望在许多类型的细胞和生物体中产生定向突变。
Zinc-finger nucleases (ZFNs) are hybrids between a nonspecific DNA-cleavage domain and a DNA-binding domain composed of Cys2His2zinc fingers. Because zinc fingers can be manipulated to recognize a broad range of sequences, these enzymes have the potential to direct cleavage to arbitrarily chosen targets. We have tested this idea by designing a pair of ZFNs that recognize a unique site in theyellow(y) gene of Drosophila. When these nucleases were expressed in developing larvae, they led to somatic mutations specifically in theygene. These somatic mosaics were observed in approximately one-half of the males expressing both nucleases. Germlineymutations were recovered from 5.7% of males, but from none of the females, tested. DNA sequences were determined and showed that all of the mutations were small deletions and/or insertions located precisely at the designed target. These are exactly the types of alterations expected from nonhomologous end joining (NHEJ) following double-strand cleavage of the target. This approach promises to permit generation of directed mutations in many types of cells and organisms.
登录
查看更多内容
影响因子：
5.6
作者：
Dreier, B;Segal, DJ;Barbas, CF
通讯作者：
Barbas, CF
DOI：
10.1073/pnas.94.11.5525
发表时间：
1997-05-27
影响因子：
11.1
作者：
Liu, Q;Segal, DJ;Barbas, CF
通讯作者：
Barbas, CF
影响因子：
10.5
作者：
GEYER, PK;CORCES, VG
通讯作者：
CORCES, VG
DOI：
10.1073/pnas.94.11.5617
发表时间：
1997-05-27
影响因子：
11.1
作者：
Isalan, M;Choo, Y;Klug, A
通讯作者：
Klug, A
果蝇中与 P 诱导的雄性重组相关的侧翼重复和缺失。
影响因子：
3.3
作者：
Preston,CR;Sved,JA;Engels,WR
通讯作者：
Engels,WR