碩士 / 國立臺灣大學 / 口腔生物科學研究所 / 105 / SP7 is a C2H2-type zinc finger transcription factor of the SP gene family and a putative master regulator of bone cell differentiation. It is identified from mouse C2C12 skeletal muscle progenitor cells, a novel zinc finger-containing transcription factor, called osterix (Osx), that is specifically expressed in all developing bones. The Osx cDNA encodes a 428-amino acid polypeptide with a predicted molecular mass of 44.7 kD.
From UCSC genome browser web site, by alignment of the SP7 gene sequence of the zebrafish and other various species (eg, medaka, mice, humans), three conserved sequences upstream of this gene (p1, p2, p3) are located. These three inter-species conserved sequences were cloned by PCR and ligated with the SP7 proximal promoter (p0) upstream of ATG into the pEGFP1 vector, which harbors the βB1-crystallin enhancer and its cognate promoter sequence that drive the reporter gene GFP in the zebrafish eye lens. The construct of proximal promoter p0 serves as the basal promoter. These constructs of chimeric enhancers-promoters (βB1-crystallin and SP7) can facilitate the early observation of GFP in the lens and the screening of transgenic lines. After I have successfully made these constructs, they were microinjected into one-cell stage zebrafish embryos. The expression patterns of EGFP were observed.
In transient transgenic assay, in F0 fish injected with p0 promoter construct, at 7 dpf (day-post-fertilization), expression of GFP was well observed in the regions of lens. Not any expression was observed in bone tissues and only some ectopic expression was observed in the notochord in few fish. In group of p1 construct, at 7 dpf, GFP expression was observed in the lens, but not in any bone tissues. However, until 84 dpf, GFP can since then be observed in the operculum, the base of pectoral fins, and ribs. In contrast, the expression patterns of p2 and p3 constructs are almost identical, but disparate from that of p1 construct. For p2 and p3 constructs, at the hatching stage (3 dpf), GFP was observed in the head mesenchyme and pectoral fins. Then at 6dpf, GFP was observed in the ventral part of notochord near caudal fins. At 13 dpf, GFP was observed in the vertebrae, in the bases of dorsal fin, anal fin, and pelvic fins, and in the ribs. The expression in these tissues continues until 1 month, and then degrades gradually.
I have screened the F0 fish and got stable lines of each construct, including p0 construct (none), p1 construct (2 lines), p2 construct (1 line), p3 construct (2 lines). The progeny of these stable lines reveals about the same expression patterns as the F0 fish, except that some ectopic expression in notochord and muscle disappeared and were not observed. And in stable lines, the expression patterns of p2 and p3 constructs are identical, insinuating the DNA duplication of these enhancer fragments have been duplicated during evolution.
Eventually, I inspect the expression of GFP during fin and scale regeneration by fin-amputation and scale-removing in p3 stable line (No.1). The experimental results revealed that no significant fluorescence of GFP was detectable in either manipulation, suggesting some enhancer-elements are still elusive in my constructs. In addition, I found the expression region in the ventral notochord near caudal fin of p2 and p3 constructs is tantamount to that of shh gene encoding a signaling molecule in previous publication. To investigate the relationship between Shh and SP7, the embryos of p3-construct F1 (No.1) zebrafish were treated with cyclopamine, an alkaloid of Shh inhibitors. The results displayed that when p3 F1 zebrafish were treated with cyclopamine at 9 dpf, the fluorescence of GFP in the caudal ventral part of notochord significantly diminished.
| Identifer | oai:union.ndltd.org:TW/105NTU05592001 |
| Date | January 2017 |
| Creators | Shi-Yi Kuo, 郭士毅 |
| Contributors | 張百恩 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 77 |
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