The Construction Of Zebrafish Endoderm And Bile Duct Lineage Tracing System

Early gastrula period is a very important period in the process of zebrafish embryonic development,destinies each layer obtained are regulated by all kinds of signals.In recent years,the ectoderm and mesoderm development process and their molecular regulation mechanism is comparative well known,but little is known about the development and regulation of endoderm.This experiment took the zebrafish as model animals to build transgenic zebrafish lines sox17:Kaede and sox17:Zebrabow,which can specific marks the endoderm during the period of the gastrula.sox17 is a transcription factor plays important role in the development of endoderm,expression lacking of sox17 will lead to the endoderm dysplasia,so take sox17 as promoter to drive color-changeable green fluorescent protein Kaede and red fluorescent protein Zebrabow to track the differentiation fate of endoderm cells is feasible.Fluorescent protein Kaede can turn to red under ultraviolet light irradiation,using this transgenic line provides a good idea for tracking differentiation destiny of single endoderm cell.Zebrabow is a newly establish system for cellular complex tracer tag based on the Cre/Loxp system,using this system can mark adjacent cells in different colors under the action of random function of Cre recombinant enzyme after Tamoxifen treatment.The application of this system can make the position of labeled cell more precise and the offspring of marked cells can express fluorescent protein constantly.This is a great progress of the cell lineage tracing technology and its establishment will create a good condition for the purpose of accurate and long-term cell lineage tracing.The liver is the most important digestive organs,playing important roles in maintaining the health of body.In recent years,deep research on molecular mechanism of liver development has been carried,but little is known about liver regeneration.Biliary duct is an important part of the liver,normal morphology and function well of bile duct is important to maintain the normal physiological function of the liver.Researches in recent years show that in certain pathological conditions the bile duct cells would transdifferentiated to form other types of liver cell to help repairing the liver.This experiment intends to build of transgenic fish Tp1bglob:Zebrabow which marks the bile duct cells and study the behavior of bile duct under condition of the liver damaged and make it clear is how it would repair the damaged liver.Construction of these three kinds of transgenic fish are all based on the I-Scel transgenic system.At first,we built three kinds of recombinant plasmid pBS-sox17-Kaede,pBS-sox17-Zbrabow and pBS-Tplbglob-Zebrabow and then injection 0.15 ng of the transgenic recombinant plasmid into the animal pole of single-cell period zebrafish embryos.After zebrafish fish injected transgenic plasmid grew to suitable stage,we use the fluorescence microscope to screen FO and select the transgenic fish whose expression pattern and fluorescence intensity are in accord with our expectation to breed and continuously extend.When extend to F2 generation,if the expression pattern and the fluorescence intensity of transgenic fish are meets our requirements,we consider the transgenic line was genetically modified and declare the transgenic.line was built successfully.In this experiment,construction of transgenic fish sox17:Kaede and Tp1bglob:Zebrabow in the region under the drive of promoter are relatively good,the fluorescence intensity can also meet our requirements.The single cell in endoderm period of sox17:Kaede turns red under the irradiation of the ultraviolet light.The transgenic lines were in line with our requirement in all aspects,and can be used for the study of zebrafish early endoderm lineage tracing.Although Tp1bglob:Zebrabow meets our requirements on the expression pattern and fluorescence intensity,the composite marking system failed to work,the bile duct cells can not be labeled in multi level,and the cause of the problem was investigated,but none is confirmed.The expression pattern in transgenic line sox17:Zebrabow appears non-specific expression in the body,but the non-specificity would have little influence to the tracing experiment.When cross with transgenic lines sox17:CreERT2,their progenies' endoderm cell can be multi-level marked after Tamoxifen treatment,and we can track the fate of these cells during the next development process.So we believe that this transgenic lines can also be used for endodermal cell lineage tracing study.