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Generation of cardiac-specific and inducible calreticulin transgenic mice.

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posted on 21.02.2013, 16:00 by Dukgyu Lee, Tatsujiro Oka, Beth Hunter, Alison Robinson, Sylvia Papp, Kimitoshi Nakamura, Wattamon Srisakuldee, Barbara E. Nickel, Peter E. Light, Jason R. B. Dyck, Gary D. Lopaschuk, Elissavet Kardami, Michal Opas, Marek Michalak

(A) The CAT-loxP-CRT transgene consisted of a CAG promoter, the CAT gene flanked with loxP-sites followed by cDNA encoding HA-tagged calreticulin. Cardiac specific cre mice express cre recombinase (MerCreMer) under the control of αMHC promoter (αMHC-Cre). Double transgenic mice were generated by breeding CAT-loxP-CRT mice with αMHC-Cre mice to produce αMHC/CAT-loxP-CRT mice. Administration of tamoxifen triggers nuclear translocation of MerCreMer and cardiac-specific Cre mediated recombination and excision of the floxed CAT gene, resulting in the expression of HA-tagged calreticulin under the control of CAG promoter (αMHC/CRT mice). αMHC/CRT mice are referred to throughout the paper as calreticulin transgenic mice (CRT-TG). Arrows and arrow heads indicate the location of primers used for PCR-driven genomic DNA analysis of transgenic mice. The location of loxP sites is indicated. CAG, CMV early enhancer/chicken β actin; HA, hemagglutinin; CAT, chloramphenicol acetyltransferase, Cre, Cre recombinase (type I topoisomerase); αMHC, α-myosin heavy chain; Mer, modified estrogen receptor. CRT, calreticulin; KDEL, ER retention signal sequence. (B) Genotyping of the αMHC-Cre, CAT-loxP-CRT, and αMHC/CAT-loxP-CRT mice was carried out by PCR-driven amplification using primers shown in Figure 1A (arrows for CAT gene and arrow heads for MerCreMer gene). (C) Western blot analysis of expression of recombinant CRT (anti-HA) and endogenous CRT (anti-CRT) after feeding tamoxifen for 3 weeks. Glyceraldehyde 3-phosphate dehydrogenase (anti-GAPDH) was used as a loading control. (D) Tissue specific expression of recombinant calreticulin (anti-HA) in CRT-TG mice and expression of endogenous calreticulin (anti-CRT) from control mouse. Sk muscle, skeletal muscle.