Endogenous zebrafish proneural Cre drivers generated by CRISPR/Cas9 short homology directed targeted integration

被引：15

作者：

Almeida, Maira P. ^{[1
,2
]}

Welker, Jordan M. ^{[1
,2
,4
]}

Siddiqui, Sahiba ^{[1
,2
]}

Luiken, Jon ^{[1
]}

Ekker, Stephen C. ^{[3
]}

Clark, Karl J. ^{[3
]}

Essner, Jeffrey J. ^{[1
,2
]}

McGrail, Maura ^{[1
,2
]}

机构：

[1] Iowa State Univ, Dept Genet Dev & Cell Biol, Ames, IA 50011 USA

[2] Iowa State Univ, Genet & Genom Interdept Grad Program, Ames, IA 50011 USA

[3] Mayo Clin, Dept Biochem & Mol Biol, Rochester, MN USA

[4] Max Planck Inst Heart & Lung Res, Dept Dev Genet 3, Bad Nauheim, Germany

来源：

SCIENTIFIC REPORTS | 2021年 / 11卷 / 01期

基金：

美国国家卫生研究院;

关键词：

TRANSCRIPTION FACTORS; BRAIN-TUMORS; GENOME; EXPRESSION; GENES; OLIG2; RECOMBINATION; NEUROGENESIS; SYSTEM; LINES;

D O I：

10.1038/s41598-021-81239-y

中图分类号：

O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];

学科分类号：

07 ; 0710 ; 09 ;

摘要：

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.

引用

页数：12

共 52 条

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