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Development of a novel system for tissue-specific and Tet-inducible shRNA expression in mice and investigation of physiological function of Junctophilin in muscle

by
Kyoung-Han Choi
M.S. Biological Science
Korea Advanced Institute of Science and Technology (KAIST)



Thesis Advisor: Jianjie Ma, Ph.D.
Graduate Program in Physiology & Integrative Biology

School of Public Health
Conference Room 258
Piscataway

Wednesday, July 28, 2010
2:00 pm


Abstract

Junctophilins (JPs) play an essential role in muscle excitation-contraction (E-C) coupling by contributing to the formation of junctional membrane complexes (JMCs). However, the lethality associated with germ-line ablation of either JP1 or JP2 prevents physiological evaluation of JPs in the maintenance of calcium homeostasis in adult striated muscles.

To investigate the physiological role of JP genes in the adult muscle, we developed a new mouse model generated by using an advanced plasmid system for RNA interference combining 1) Cre-loxP-mediated recombination for a tissue-specific expression, 2) tTS and rtTA-mediated tetracycline (or doxycycline)-on system for a tight control and robust induction of expression and 3) Use of mir-shRNA with Dicer- and Drosha-RNase processing sites mimicking natural micro-RNA for an efficient production of small-interfering RNA (siRNA). The mir-shRNA expression cassette derived by a tetracycline-responsive CMV promoter is not functional until an interrupting GFP expression cassette flanked by loxP sites is excised by Cre-mediated recombination. Thus, tissue- or lineage- specific expression of the shRNA can be achieved by the use of appropriate inducer mice expressing tissue-specific Cre.

In transgenic mice, the expression cassette for mir-shRNA targeting JP mRNAs (mirJP shRNA) was efficiently activated by the excision of GFP expression cassette in skeletal muscles by skeletal muscle-specific Cre. While this system tightly regulated leaky expression of mirJP shRNA in the absence of doxycycline, it induced robust knockdown of both JP1 and JP2 by treatment with doxycycline. Moreover, knockdown of JPs significantly reduced store-operated Ca2+ entry (SOCE) and induced abnormal structures of triad junction in the skeletal muscle, indicating that JPs play an important role in both the regulation of intracellular Ca2+ homeostasis and the formation of JMCs. Furthermore, the suppression of the mirJP shRNA expression by doxycycline withdrawal restored SOCE activity and morphology of triad junction, indicating that the system is fully reversible in animal model in a doxycycline-dependent manner.
Biochemical and phenotypic assays demonstrate that this system is a very useful tool to study gene function with high potential of multiple applications to down-regulate specific target gene in a tissue specific and inducible manner in both in vitro and in vivo models.


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