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Cryopreservation of Mouse Embryos by Ethylene Glycol-Based Vitrification
JoVE 3155 11/18/2011

3xFLAG-dCas9 expressing mice for enChIP analysis


title201910

3xFLAG-dCas9 expressing mice for enChIP analysis

C57BL/6-Gt(ROSA)26Sor<tm1(CAG-3XFLAG/dCas9,-EGFP)Hfuj> (RBRC10188)
C57BL/6-Gt(ROSA)26Sor<tm1.1(CAG-3XFLAG/dCas9,-EGFP)Hfuj> (RBRC10189)
C57BL/6-Gt(ROSA)26Sor<tm1.2(CAG-3XFLAG/dCas9)Hfuj> (RBRC10190)

Fig_201910

Courtesy of Hodaka Fujii, M.D., Ph.D.

Structures of expression cassettes of 3xFLAG-dCas9 and a scheme of enChIP analysis using transgenic mice expressing 3xFLAG-dCas9

 

Molecular interaction analysis is essential to understand molecular mechanisms of genomic functions (transcription, epigenetic regulation, etc.). Engineered DNA-binding molecule-mediated chromatin immunoprecipitiation (enChIP) is a novel ChIP method with combination of CRISPR/Cas9 system [1,2].

This enables to identify associated proteins, DNA, RNA or other molecules with genomic regions of interest in a non-biased manner. The developers of enChIP analysis (Dr. Fujii and his colleagues) established useful transgenic mouse lines for enChIP analysis in primary mouse cells [3]. These strains express catalytically inactive form of Cas9 proteins of Streptococcus pyogenes which is tagged with 3xFLAG (3xFLAG-dCas9) under the control of CAG promoter in the ROSA26 locus.

The experiment flow is roughly as follows: (1) design of guide RNA (gRNA) to a specific genomic locus; (2) isolation of objective cells from 3xFLAG-dCas9 expressing mice; (3) transfection/transduction of target specific gRNA for formation of gRNA and 3xFLAG-dCas9 complexes inside cells; (4) purification of the complexes by immunoprecipitiation; (5) identification of associated molecules by mass spectrometry, next-generation sequencing, and so on. The data of enChIP analysis will elucidate the unknown mechanisms of biological phenomenon and disease onset.

 

Depositor : Hodaka Fujii, M.D., Ph.D.
Department of Biochemistry and Genome Biology,
Hirosaki University Graduate School of Medicine
Strain name : C57BL/6-Gt(ROSA)26Sor<tm1(CAG-3XFLAG/dCas9,-EGFP)Hfuj>
(3xFLAG-dCas9 or fluorescent reporter inducible expression mice by Cre/loxP and Flp/FRT system)
RBRC No. : RBRC10188
Strain name : C57BL/6-Gt(ROSA)26Sor<tm1.1(CAG-3XFLAG/dCas9,-EGFP)Hfuj>
(3xFLAG-dCas9 and fluorescent reporter expression mice)
RBRC No. : RBRC10189
Strain name : C57BL/6-Gt(ROSA)26Sor<tm1.2(CAG-3XFLAG/dCas9)Hfuj>
(3xFLAG-dCas9 constitutive expression mice)
RBRC No. : RBRC10190
References : [1] Fujita T, Fujii H.
Efficient isolation of specific genomic regions and identification of associated proteins by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using CRISPR.
Biochem Biophys Res Commun.; 439(1):132-6, 2013.
[2] Fujita T, Asano Y, Ohtsuka J, Takada Y, Saito K, Ohki R, Fujii H.
Identification of telomere-associated molecules by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP).
Sci Rep.; 3:3171, 2013.
[3] Fujita T, Kitaura F, Oji A, Tanigawa N, Yuno M, Ikawa M, Taniuchi I, Fujii H.
Transgenic mouse lines expressing the 3xFLAG-dCas9 protein for enChIP analysis.
Genes Cells.; 23(4):318-325, 2018.

 

Ocrober 2019
Saori Mizuno, Ph.D.
Contact: Experimental Animal Division, RIKEN BioResource Research Center (animal@brc.riken.jp)
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