使綠螢光能活體標記於類心臟之DNA新組合及其製備的方法(Method for producing heart-specific fluorescence of non-human eukaryotic animals )
  ‧ 專利名稱 使綠螢光能活體標記於類心臟之DNA新組合及其製備的方法(METHOD FOR PRODUCING HEART-SPECIFIC FLUORESCENCE OF NON-HUMAN EUKARYOTIC ANIMALS )
  ‧ 專利證書號 US 7,355,095 B2
  ‧ 專利權人 國立臺灣大學
  ‧ 專利國家
    (申請日)
美國 (2002/02/22)
美國 (2004/12/22)
  ‧ 發明人 蔡懷楨, 黃秋茹, 蕭崇德,
 
技術摘要:
A method of expressing in vivo heart-specific fluorescence in transgenic line of zebrafish is developed, which provides a research model for studying heart-related gene functions and performing gene therapies in the future. The method comprises the following step. A fluorescent protein gene is integrated into the genome of a non-human eukaryotic animal. In a preferred embodiment, a gene encoding GFP is transferred into the genome of a zebrafish. The transgenic process comprises the following steps. Firstly, the genomic DNA of zebrafish larvae are extracted and cut with a restriction enzyme. Then, the DNA fragments are ligated with adaptors, Pad1 and PR-SpeI. After ligation, PCR is performed twice to amplify the target DNA fragment. The amplified fragment is subjected to gene sequencing steps for determing the nucleotide sequence, which is the 5′ region of zebrafish cmlc2 gene. Subsequently, a plasmid is constructed. This plasmid construct includes the upstream regulatory region, the exon 1, the intron 1, and the exon 2 of cmlc2 gene, cDNA of GFP, wherein the cmlc2 gene and GFP cDNA form a cassette, and inverted terminal repeats from adeno-associated virus are flanked at both sides of this cassette. The plasmid construct is linearized and microinjected into one-celled zebrafish fertilized eggs. Lastly, the heart-specific fluorescent expressed zebrafish are selected and the germline-transmitting transgenic strain is generated.



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