| Summary: | 碩士 === 國立臺灣海洋大學 === 生物科技研究所 === 99 === Liver and pancreas arise from common progenitors in the posterior foregut endoderm. In recent years, many scientists use master regulator (transcription factor) like Pdx1 and Ptf1a that they successfully convert liver into pancreas. Previously studies,Pdx1-VP16(modified form of Pdx1, pancreatic and duodenal homeobox 1) cause conversion of liver cells to pancreatic cell fate(endocrine and exocrine cell fate).And the other one, Ptf1a-VP16 (modified form of Ptf1a, pancreas specific transcription factor, 1a) also can cause transdifferentiation from liver to pancreas (only exocrine pancreatic cell fate).But the function of Pdx1 and Ptf1a co-expression in liver cells is unclear. And unmodified Pdx1,Ptf1a do not convert liver to pancreas. Therefore, the aim of this experiment is to determine whether Pdx1 and Ptf1a are able to synergistically promote liver to pancreas conversion in zebrafish. The experiment use liver specific promoter LFABP to drive Pdx1 and Ptf1a expressions in zebrafish. Two experiment construct in use: P-P(Pdx1-Ptf1a) construct and CRC(Tet-Off) construct. Each of them are quite different in strategy design. P-P construct can be induced by adding Doxycycline or RU486. They are used to control Pdx1-VP16 or Ptf1a-VP16 or both expression independently.CRC construct’s strategy are used to produce positive feedback chain reaction. And I find that the other factor named Rbpj is included in PTF1 complex, which is important for early pancreas
development. Pdx1,Ptf1a,Rbpj expression can be increased to the maximum in CRC construct. Using transient experiment to know about P-P(Pdx1-Ptf1a) construct and CRC construct ability. Each construct is microinjected into zebrafish embryo at one cell stage. RNA whole mount in situ hybridization (trypsin, exocrine pancreas marker) analysis found that two systems both can cause ectopic expression of exocrine cells(trypsin) in liver. The ectopic expressions are at left side(liver),yolk, Tail, etc. Using LIP Fluorescent fishes also showed similar results at in
situ hybridization. However, the survival rate is quite low, especially only 20%~30% in CRC experiment, and it may be resulted from two reason:1) Those factors mis-express in other tissues .2)The failure of transdifferentiation event cause develop abnormally in early liver cells. In this study, I successfully cause transdifferentiation in zebrafish. However, it have to do much further study to understand the gene network of those
genes in zebrafish.
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