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Video protocol

Cryopreservation of Mouse Embryos by Ethylene Glycol-Based Vitrification

JoVE 3155 11/18/2011

Green-to-red fluorescent-convertible Cre-reporter mice


title201810

Green-to-red fluorescent-convertible Cre-reporter mice

C57BL/6N-Gt(ROSA)26Sor<tm1(CAG-EGFP/DsRed)Utr>/Rbrc (RBRC04874)

figure A
figure B

Courtesy of Fumihiro Sugiyama, Ph.D.

(A) Cre-recombination strategy in R26GRR mice.
Cre-reporter cassette is knocked into ROSA26 locus. Prior to Cre-recombination, only first reporter EGFP signal is emitted. Following Cre recombination, EGFP-excision is occurred and second reporter tdsRed signal is emitted. Both endogenous ROSA26 promoter and exogenous CAG promoter achieve ubiquitous and constitutive expression of each reporter gene.
(B) Cre-dependent site specifically detection of tdsRed signals.
As mouse insulin1 (Ins1) are expressed in pancreatic β-cells, Ins1-Cre driver mice are expected to express Cre recombinase at same site. In heterozygous R26GRR (R26GRR+/KI) mice which do not have Cre recombinase, EGFP and no tdsRed emission were detected in whole pancreas. On the other hand, in R26GRR/Ins1-Cre F1 (R26GRR+/KI; Ins1-cre+/Tg) mice, red dot spots were detected over the pancreas. From another experiments, it was revealed that these tdsRed signals were correspond with Ins1-expressing sites.

 

Cre recombinase derived from P1 bacteriopharge specifically recognize short DNA sequences (loxP site) and cause inducible genome alternation. The Cre/loxP system have been widely used to study gene function in vivo as conditional knockout mice. Successful Cre-mediated recombination in mice depends on accurate Cre expression in Cre-driver mice. R26GRR mice are knock-in mice from C57BL/6N ES cells, in which Cre-reporter cassette into ROSA26 locus. As Cre-reporter mice, R26GRR mice are useful for evaluation of Cre expression pattern in Cre-diver mice (1). R26GRR mice make possible to monitor Cre-mediated recombination by dual fluorescent signal with different wavelengths (EGFP and tdsRed). Depositor of R26GRR mice (Sugiyama) and colleagues already utilized R26GRR mice to characterize some novel site-specific Cre-driver mice, which they generated (2, 3, 4). They revealed that R26GRR mice show green fluorescence in all organs examined before and red fluorescence in only predictable site after Cre recombination.

 

Depositor : Laboratory Animal Resource Center, University of Tsukuba
and RIKEN BioResource Center
Strain name : C57BL/6N-Gt(ROSA)26Sor<tm1(CAG-EGFP/DsRed)Utr>/Rbrc
RBRC No. : RBRC04874
References : [1] Hasegawa Y, Daitoku Y, Sekiguchi K, Tanimoto Y, Mizuno-Iijima S, Mizuno S, Kajiwara N, Ema M, Miwa Y, Mekada K, Yoshiki A, Takahashi S, Sugiyama F, Yagami K. Novel ROSA26 Cre-reporter knock-in C57BL/6N mice exhibiting green emission before and red emission after Cre-mediated recombineation. Exp Anim.; 62(4): 295-304, 2013.
[2] Hasegawa Y, Daitoku Y, Mizuno S, Tanimoto Y, Mizuno-Iijima S, Matsuo M, Kajiwara N, Ema M, Oishi H, Miwa Y, Mekada K, Yoshiki A, Takahashi S, Sugiyama F, Yagami K. Generation and characterization of Ins1-cre-driver C57BL/6N for exclusive pancreatic beta cell-specific Cre-loxP recombination. Exp Anim.; 63(2): 183-191, 2014.
[3] Hasegawa Y, Hoshino Y, Ibrahim AE, Kato K, Daitoku Y, Tanimoto Y, Ikeda Y, Oishi H, Takahashi S, Yoshiki A, Yagami K, Iseki H, Mizuno S, Sugiyama F. Generation of CRISPR/Cas9-mediated bicistronic knock-in ins1-cre driver mice. Exp Anim.; 65(3): 319-327, 2016.
[4] AI-Soudy AS, Nakanishi T, Mizuno S, Hasegawa Y, Shawki HH, Katoh MC, Basha WA, Ibrahim AE, EI-Shemy HA, Iseki H, Yoshiki A, Hiromori Y, Nagase H, Takahashi S, Oishi H, Sugiyama F. Germline recombineation in a novel cre transgenic line, Prl3b1-Cre mouse. Genesis; 54(7):389-397, 2016.

 

October 2018
Contact: Saori Mizuno, Ph.D.
Experimental Animal Division, RIKEN BioResource Research Center
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