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

目前斑馬魚中要以弁鈳鄍

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.