Use of RecA fusion proteins to induce genomic modifications in zebrafish.
Use of RecA fusion proteins to induce genomic modifications in zebrafish.
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DOI：
10.1093/nar/gkq1363
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发表时间：
2011-05
影响因子：
14.9
通讯作者：
Essner JJ
中科院分区：
文献类型：
--
作者：
Liao HK;Essner JJ
细菌重组酶RecA在双链断裂（DSB）修复期间在单链（ss）DNA上形成核酸-蛋白质细丝，其有效地经历同源性搜索并与互补DNA序列配对。我们利用RecA-DNA丝的配对活性将生化活性拴系到特定的染色体位点。测试了具有嵌合RecA蛋白的不同细丝在单细胞阶段注射后诱导斑马鱼黄金位点杂合性丢失的能力。含有核定位信号（NLS）的RecA与Gal 4的DNA结合结构域之间的融合蛋白（NLS-RecA-Gal 4）显示出最大的活性。我们的研究结果表明，短至60个核苷酸的互补ssDNA丝与NLS-RecA-Gal 4蛋白包被能够导致约3%的注射胚胎的杂合性丢失。我们证明，病变的F0代斑马鱼的10.9%的大染色体缺失传递给后代。线性DNA与NLS-RecA-Gal 4 DNA细丝的共注射促进DNA插入靶向基因组位置。我们的数据支持NLS-RecA-Gal 4 DNA细丝结合到染色质上的互补靶位点并阻止DNA复制叉，从而导致DNA DSB的模型。
The bacterial recombinase RecA forms a nucleic acid-protein filament on single-stranded (ss) DNA during the repair of double-strand breaks (DSBs) that efficiently undergoes a homology search and engages in pairing with the complementary DNA sequence. We utilized the pairing activity of RecA–DNA filaments to tether biochemical activities to specific chromosomal sites. Different filaments with chimeric RecA proteins were tested for the ability to induce loss of heterozygosity at the golden locus in zebrafish after injection at the one-cell stage. A fusion protein between RecA containing a nuclear localization signal (NLS) and the DNA-binding domain of Gal4 (NLS-RecA-Gal4) displayed the most activity. Our results demonstrate that complementary ssDNA filaments as short as 60 nucleotides coated with NLS-RecA-Gal4 protein are able to cause loss of heterozygosity in ∼3% of the injected embryos. We demonstrate that lesions in ∼9% of the F0 zebrafish are transmitted to subsequent generations as large chromosomal deletions. Co-injection of linear DNA with the NLS-RecA-Gal4 DNA filaments promotes the insertion of the DNA into targeted genomic locations. Our data support a model whereby NLS-RecA-Gal4 DNA filaments bind to complementary target sites on chromatin and stall DNA replication forks, resulting in a DNA DSB.
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影响因子：
14.9
作者：
Feitsma H;Akay A;Cuppen E
通讯作者：
Cuppen E
DOI：
10.1073/pnas.0908247106
发表时间：
2009-11-03
影响因子：
11.1
作者：
Petzold, Andrew M.;Balciunas, Darius;Ekker, Stephen C.
通讯作者：
Ekker, Stephen C.
影响因子：
64.5
作者：
Boddy, MN;Gaillard, PHL;Russell, P
通讯作者：
Russell, P
影响因子：
3.2
作者：
Keller, M;Harbottle, RP;Miller, AD
通讯作者：
Miller, AD
DOI：
10.1038/nbt1290-1268
发表时间：
1990-12-01
期刊：
BIO-TECHNOLOGY
影响因子：
--
作者：
LIU, ZJ;MOAV, B;HACKETT, PB
通讯作者：
HACKETT, PB