Functional visualization and disruption of targeted genes using CRISPR/Cas9-mediated eGFP reporter integration in zebrafish.
Functional visualization and disruption of targeted genes using CRISPR/Cas9-mediated eGFP reporter integration in zebrafish.
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DOI：
10.1038/srep34991
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发表时间：
2016-10-11
影响因子：
4.6
通讯作者：
Kawahara A
中科院分区：
文献类型：
--
作者：
Ota S;Taimatsu K;Yanagi K;Namiki T;Ohga R;Higashijima SI;Kawahara A
CRISPR/Cas9复合体由一个引导RNA(GRNA)和Cas9核酸酶组成，在各种生物中进行基因组修饰是有用的。最近，CRISPR/Cas9介导的一种报告基因的位置特异性整合，其中包含以Mbait-gRNA为靶点的Mbait序列，HSP70启动子和EGFP基因，使目标基因的表达得以可视化。然而，目前还没有确定这两个目标等位基因上的报告整合是否会导致斑马鱼功能丧失的表型。在这项研究中，我们将Mbait-hs-EGFP报告基因插入到pax2a基因中，因为pax2a的破坏导致斑马鱼中脑-后脑边界(MHB)的丢失。在杂合子Tg[pax2a-hs：EGFP]胚胎中，MHb形成正常，EGFP表达概括了内源性pax2a的表达，包括MHb。我们观察到纯合子Tg[pax2a-hs：EGFP]胚胎中MHB的丢失。此外，我们成功地将Mbait-hs-EGFP报告基因整合到一个未鉴定的基因epdr1中。在杂合子TG[epdr1-hs：EGFP]胚胎中，EGFP的表达与epdr1的表达重叠，而在纯合子TG[epdr1-hs：EGFP]胚胎的MHB中，EGFP阳性细胞的分布是混乱的。我们认为，Mbait-hs-EGFP报告基因的位点特异性整合是研究基因表达谱和功能丧失表型的有效方法。
The CRISPR/Cas9 complex, which is composed of a guide RNA (gRNA) and the Cas9 nuclease, is useful for carrying out genome modifications in various organisms. Recently, the CRISPR/Cas9-mediated locus-specific integration of a reporter, which contains the Mbait sequence targeted using Mbait-gRNA, the hsp70 promoter and the eGFP gene, has allowed the visualization of the target gene expression. However, it has not been ascertained whether the reporter integrations at both targeted alleles cause loss-of-function phenotypes in zebrafish. In this study, we have inserted the Mbait-hs-eGFP reporter into the pax2a gene because the disruption of pax2a causes the loss of the midbrain-hindbrain boundary (MHB) in zebrafish. In the heterozygous Tg[pax2a-hs:eGFP] embryos, MHB formed normally and the eGFP expression recapitulated the endogenous pax2a expression, including the MHB. We observed the loss of the MHB in homozygous Tg[pax2a-hs:eGFP] embryos. Furthermore, we succeeded in integrating the Mbait-hs-eGFP reporter into an uncharacterized gene epdr1. The eGFP expression in heterozygous Tg[epdr1-hs:eGFP] embryos overlapped the epdr1 expression, whereas the distribution of eGFP-positive cells was disorganized in the MHB of homozygous Tg[epdr1-hs:eGFP] embryos. We propose that the locus-specific integration of the Mbait-hs-eGFP reporter is a powerful method to investigate both gene expression profiles and loss-of-function phenotypes.
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影响因子：
7
作者：
Auer TO;Duroure K;De Cian A;Concordet JP;Del Bene F
通讯作者：
Del Bene F
DOI：
10.1016/j.tig.2008.08.007
发表时间：
2008-11
期刊：
Trends in genetics : TIG
影响因子：
--
作者：
McVey M;Lee SE
通讯作者：
Lee SE
影响因子：
2.4
作者：
Ansai S;Kinoshita M
通讯作者：
Kinoshita M
影响因子：
4.6
作者：
Hisano Y;Sakuma T;Nakade S;Ohga R;Ota S;Okamoto H;Yamamoto T;Kawahara A
通讯作者：
Kawahara A
DOI：
10.1242/dev.115584
发表时间：
2014-12
期刊：
Development (Cambridge, England)
影响因子：
--
作者：
Irion U;Krauss J;Nüsslein-Volhard C
通讯作者：
Nüsslein-Volhard C