誘導生成反義股來抑制斑馬魚心臟肌鈣蛋白的表現

Knock Down the Expression of Zebrafish Cardiac Troponin C in Heart by Conditionally Generating Antisense Strand in vivo

王嘉琳(Chia-Lin Wang)

指導教授 ： 蔡懷楨

國立臺灣大學/生命科學院/漁業科學研究所/碩士(2004年)

https://doi.org/10.6342/NTU.2004.10026

摘要

目前斑馬魚中要以弁鈳鄍

關鍵字

肌鈣蛋白 ； 心臟 ； 斑馬魚 ； 反義股

並列摘要

The tetracycline-controlled myocardium expression system was used to study zebrafish cardiac troponin C (cTnC), a calcium binding protein specifically expressed in heart. GFP reporter and antisense strand of cTnC were constructed on both side of the bi-directional promoter, which was response to the doxycycline (Dox)-induced transactivator driven by a heart –specific promoter of cardiac myosin light chain 2. GFP reporter gene was specifically expressed in the heart of transgenic fish F1 when 10μg/ml of Dox was continuously treated the 12-hpf embryos. We found that the average heart-beating rate of cTnC-antisense transgenic fish was significantly slower than that of the control fish at 6-dpf (150.2 vs. 193.7 beats per min) and 12-dpf (127.8 vs. 168.4 beats per min). The transcription of antisese mRNA of cTnC was detected by RT-PCR at 6-dpf. Furthermore, ventricle morphology was measured from video images of the 6-dpf and 12-dpf embryos in order to compare the difference in term of ventricular function between the control and cTnC-antisense transgenic fish. The end-diastolic and end-systolic volumes of the ventricle of the latter fish were increased, while the ventricular ejection fractions were decreased. Embryos that were injected with low dosage (0.03375ng) of cTnC-morpholino (MO) photocopied the defects of the cTnC-antisense transgenic fish. An asymmetric heart-beat between atrium and ventricle, a syndrome that is similar to the human incomplete atrio- ventricular blocking disease, was observed in few induced hearts (1.3%). These evidences demonstrate that the heart-specific protein is capable of being partially inhibited to translate in the transgenic zebrafish, suggesting this tet-on system can be potentially applied in studying heart disease.

並列關鍵字

Heart ； tet-on system ； cTnC ； troponin C ； zebrafish

參考文獻

Lee W.H. (2003) Conditional expression in the zebrafish heart with tet-on regulatory system. Master thesis, National Taiwan University, Taipei, Taiwan.

Dodd A., Chambers S.P. and Love D.R. (2004) Short interfering RNA-mediated gene targeting in the zebrafish. Federation of European Biochemical Societies 561, 89-93.

Gillis T.E. and Tibbits G.F. (2002) Beating the cold: the functional evolution of troponin C in teleost fish. Comparative Biochemistry and Physiology 132A, 763-772.

Gossen M. and Bujard H. (1995) Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proceedings of the National Academy of Sciences of the United States of America 89, 5547-5551.

Gossen M., Freundlieb S. and Bender G. (1995) Transcriptional activation by tetrayclines in mammalian cells. Science 268, 1766-1769.