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Impaired effect of BHC80 gene knock-down on the cardiac development in zebrafish / 生理学报
Acta Physiologica Sinica ; (6): 547-552, 2013.
Article in Chinese | WPRIM | ID: wpr-297538
ABSTRACT
The effect of BHC80 (a component of BRAF-HDAC complex) on development was not well studied, because BHC80 gene knock-out mice died in one day after birth. Interestingly, zebrafish embryos can live, even if their important organs like cardiac system has severe dysfunction, as 25%-40% O2 are supplied through their skin. Therefore, a model of BHC80 gene knock-down zebrafish embryos was established to explore the effect of BHC80 on the early embryonic development. BHC80-morpholino antisense oligonucleotides 2 (BHC80-MO2) was designed and injected into zebrafish embryos to interrupt the correct translation of BHC80 mRNA at one or two cells stage, which was proved by RT-PCR analysis. Two control groups, including non-injection group and control-MO (con-MO) injection group, and four different doses of BHC80-MO2 injection groups, including 4 ng, 6 ng, 8 ng and 10 ng per embryo were set up. The embryonic heart phenotype and cardiac function were monitored, analyzed and compared between con-MO and BHC80-MO2 groups by fluorescence microscope in vmhcgfp transgenic zebrafish which express green fluorescent protein in ventricle. The results showed that BHC80-MO2 microinjection effectively knocked down the BHC80 gene expression, because the BHC80-MO2 group emerged a new 249 bp band which reduced 51 bp compared to 300 bp band of con-MO group in RT-PCR analysis, and the 51 bp was the extron 10. The abnormal embryo rate rose with the increase of BHC80-MO2 dosage. The proper BHC80-MO2 injection dosage was 8 ng per embryo, as minor embryos had abnormal phenotype in 4 ng and 6 ng per embryo groups and most embryos died in 10 ng per embryo group. BHC80-MO2 embryos exhibited abnormal cardiac phenotype, including imbalance of the proportion of heart ventricle to atrium, incomplete D-loop, even tubular heart, slow heart rates and cardiac dysfunction. The results from a model of BHC80 gene knock-down zebrafish embryos show that the abnormal cardiac phenotype and cardiac dysfunction of BHC80-MO2 embryos may be one of the probable reasons for the BHC80 gene knock-out mice death, which would provide a good research model to clarify the mechanism of cardiac development.
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Full text: Available Index: WPRIM (Western Pacific) Main subject: Zebrafish / RNA, Messenger / Embryology / Oligonucleotides, Antisense / Mice, Knockout / Gene Expression Regulation, Developmental / Zebrafish Proteins / Embryonic Development / Gene Knockdown Techniques / Genetics Limits: Animals Language: Chinese Journal: Acta Physiologica Sinica Year: 2013 Type: Article

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Full text: Available Index: WPRIM (Western Pacific) Main subject: Zebrafish / RNA, Messenger / Embryology / Oligonucleotides, Antisense / Mice, Knockout / Gene Expression Regulation, Developmental / Zebrafish Proteins / Embryonic Development / Gene Knockdown Techniques / Genetics Limits: Animals Language: Chinese Journal: Acta Physiologica Sinica Year: 2013 Type: Article