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KMID : 0613820170270060617
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Journal of Life Science
2017 Volume.27 No. 6 p.617 ~ p.622
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Expression of Human Cytochrome b5 in Zebrafish
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Han Se-Mi
Yoo Min
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Abstract
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In this study, we sought to develop an effective cloning system by which human cytochrome b5 (cyt b5) is introduced and expressed in zebrafish. First, the 414 bp human cyt b5 gene was amplified from RNA extracts of HeLa cells using RT-PCR, and the amplicon was subsequently sequenced to confirm that it was intact. Next, cyt b5 was cloned into the pEGFP-N3 vector, which also encodes a fluorescent gene. One-cell stage zebrafish embryos were microinjected with the recombinant vector containing the cyt b5 gene. Fluorescence microscopy confirmed high expression of the fluorescent gene in the injected fry compared to the non-fluorescent control fry. Finally, we extracted RNA from the injected fry and performed RT-PCR to determine whether the human cyt b5 gene is expressed in the transgenic zebrafish. Sequencing analysis further confirmed that the cloned human cyt b5 gene was intact. The transgenic zebrafish model produced in this study will be a useful tool to study therapeutic approaches to cure various diseases related to the deficiency of functional human cyt b5 as well as tools for cloning useful genes in fish.
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KEYWORD
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Cytochrome b5, HeLa cell, methemoglobinemia, pEGFP-N3, zebrafish
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