Endogenous zebrafish proneural Cre drivers generated by CRISPR/Cas9 short homology directed targeted integration.
Endogenous zebrafish proneural Cre drivers generated by CRISPR/Cas9 short homology directed targeted integration.
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DOI：
10.1038/s41598-021-81239-y
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发表时间：
2021-01-18
影响因子：
4.6
通讯作者：
McGrail M
中科院分区：
文献类型：
--
作者：
Almeida MP;Welker JM;Siddiqui S;Luiken J;Ekker SC;Clark KJ;Essner JJ;McGrail M
我们之前报道了利用CRISPR/Cas9和短同源区域在斑马鱼和人类细胞中高效、精确地靶向整合报告DNA。在这里，我们应用该策略分离出空间和时间限制性表达模仿内源性基因的斑马鱼Cre重组酶驱动因子。一个具有48 bp同源臂的2A-Cre重组酶转基因基因被靶向到原基因ascl1b、olig2和neurod1中。我们观察到高的种系传播率从10%到100%不等（2/20 olig2; 1/5 neurod1; 3/3 ascl1b）。转基因系Tg(ascl1b-2A-Cre)is75、Tg(olig2-2A-Cre)is76和Tg(neurod1-2A-Cre)is77在预期的前膜细胞群中表达功能性Cre重组酶。2A-CreERT2的体细胞靶向进入神经d1导致神经系统中他莫昔芬反应性重组。结果表明，Cre重组酶的表达受靶基因的天然启动子和调控元件的驱动。该方法提供了一种简单、高效、经济的方法来生成细胞类型特异性的斑马鱼Cre和CreERT2驱动程序，克服了启动子bac和转座子介导的转基因相关的挑战。
We previously reported efficient precision targeted integration of reporter DNA in zebrafish and human cells using CRISPR/Cas9 and short regions of homology. Here, we apply this strategy to isolate zebrafish Cre recombinase drivers whose spatial and temporal restricted expression mimics endogenous genes. A 2A-Cre recombinase transgene with 48 bp homology arms was targeted into proneural genes ascl1b, olig2 and neurod1. We observed high rates of germline transmission ranging from 10 to 100% (2/20 olig2; 1/5 neurod1; 3/3 ascl1b). The transgenic lines Tg(ascl1b-2A-Cre)is75, Tg(olig2-2A-Cre)is76, and Tg(neurod1-2A-Cre)is77 expressed functional Cre recombinase in the expected proneural cell populations. Somatic targeting of 2A-CreERT2 into neurod1 resulted in tamoxifen responsive recombination in the nervous system. The results demonstrate Cre recombinase expression is driven by the native promoter and regulatory elements of the targeted genes. This approach provides a straightforward, efficient, and cost-effective method to generate cell type specific zebrafish Cre and CreERT2 drivers, overcoming challenges associated with promoter-BAC and transposon mediated transgenics.
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影响因子：
3.7
作者：
Hozumi S;Hirabayashi R;Yoshizawa A;Ogata M;Ishitani T;Tsutsumi M;Kuroiwa A;Itoh M;Kikuchi Y
通讯作者：
Kikuchi Y
影响因子：
4.6
作者：
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影响因子：
2.7
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影响因子：
4.6
作者：
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通讯作者：
Kawahara A
影响因子：
2.7
作者：
Kani, Shuichi;Bae, Young-Ki;Hibi, Masahiko
通讯作者：
Hibi, Masahiko