Studying on the Non-specific and Tissue-specific Genes of Model Fish

• Main Authors: Long Shyan Horng, 洪龍賢
• Other Authors: Huai Jen Tsai
• Format: Others
• Language: zh-TW
• Published: 2001
• Online Access: http://ndltd.ncl.edu.tw/handle/11085635500293538270

碩士 === 國立臺灣大學 === 漁業科學研究所 === 89 === A green fluorescent protein (GFP) cDNA flanked by inverted terminal repeats (ITR) of adeno-associated virus was constructed. The construct sharply improved the efficiency and specificity of the transient expression of genes driven by general promoters (medaka β-actin) and muscle-specific promoter (zebrafish α-actin) in transgenic medaka. In addition, treatment with ITR sequence-containing constructs resulted in a dramatic increase in the number of embryos showing uniform GFP-expression at F0. Of the GFP-positive embryos, showed homogenous GFP-expression for the derivative constructs of theα-actin andβ-actin promoters, respectively. As a result of uniform GFP-expression, green fluorescence in founders was a) extended for an entire lifetime without degradation, and b) transmitted as a genetic trait to F1 and F2 progeny of some transgenic lines via Mendelian inheritance. A Southern blot analysis revealed a random integration of the transgene into the genome of founders and progeny in both head-to-tail and tail-to-tail concatemerization patterns. Interestingly, some transgenic medaka with uniform and strong fluorescence could be visually noticeable to the unaided eye. On the other way, in chapter 2 I focused my research on the zebrafish TnT 1 gene. At first, what is called Troponin complex? One of muscle structural proteins, is composed of three subunits: Troponin C（TnC）, Troponin T（TnT） and Troponin I（TnI）. Based on the expressional locations, TnT is subdivided into slow muscle type (TnT1), cardiac muscle type (TnT2) and fast muscle type (TnT3). Although heterogeneities of TnT have been studied in mammal and chicken, extremely few fish TnT genes are known in terms of gene structure and regulation. We cloned a full-length of zebrafish TnT1 (ZF-TnT1) cDNA from 24 hpf embryos. This ZF-TnT1 cDNA was around 1.4 kb and encoded 290 amino acid residues, in which glutamic acids were rich in the N-terminus. Compared with chicken TnT2, salmon TnT1, zebrafish TnT3 and salmon TnT3, ZF-TnT1 shared 84, 65, 60 and 54 % amino acid identity, respectively. Using whole-mount in situ hybridization, I found that ZF-TnT1 transcripts were first detectablen at 12 hpf embryos, increased substantially at 24 hpf, and then declined gradually until 48 hpf stage. ZF-TnT1 signals were totallyundetectable in the trunk muscle of 48 hpf embryos, but, interestingly enough, ZF-TnT1 transcripts were found in fin buds and head muscle. Using cryosection, I found that ZF-TnT1 transitional expression in slow and fast muscles. These all evidences strongly suggest that ZF-TnT1 is a muscle and stage specific TnT isoform, which may be involved in the early development of muscle cells.