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This dissertation, "Double-stranded RNA Induced Gene Silencing of Neuropeptide Genes in Sand Shrimp, Metapenaeus Ensis and Development of Crustacean Primary Cell Culture" by Haoji, Guan, 關浩基, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation This dissertation, "Double-stranded RNA Induced Gene Silencing of Neuropeptide Genes in Sand Shrimp, Metapenaeus Ensis and Development of Crustacean Primary Cell Culture" by Haoji, Guan, 關浩基, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract of thesis entitled DOUBLE-STRANDED RNA INDUCED GENE SILENCING OF NEUROPEPTIDE GENES IN SAND SHRIMP, METAPENAEUS ENSIS AND DEVELOPMENT OF CRUSTACEAN PRIMARY CELL CULTURE Submitted by Haoji Guan for the degree of Master of Philosophy at The University of Hong Kong in August 2006 The technique using RNA interference (RNAi) to study gene function in different organisms has emerged as a powerful tool in the field of reverse genetics. Although the molecular aspects of RNAi have been studied primarily in insects, there are hints that similar RNAi might occur in the shrimp Metapenaeus ensis as well. The eyestalks of shrimp produce a group of neurohormones that belong to crustacean hyperglycemic hormone (CHH) / molt inhibiting hormone (MIH) / gonad inhibiting hormone (GIH) family. These hormones control diverse processes such as molting and reproduction in M. ensis. MIH is produced in the neurosecretory cells and inhibits molting by suppressing the production of the molting hormone (ecdysone) produced from the Y-organ. GIH plays an inhibitory role in vitellogenesis since GIH mRNA transcript in the eyestalk decreases in the initial phase of gonad maturation and increases towards the end of maturation. In this report, an RNA interference (RNAi) technique was developed to study the function the putative MIH and GIH in the sand shrimp M. ensis. Double stranded RNA for GIH caused the in vitro gene silencing of GIH in the nerve cord in as early as 12 hr. In vivo injection of GIH dsRNA into the female led to the decline of GIH transcript level in the nerve cord, but the vitellogenin (MeVg2) transcript level in the hepatopancreas was increased. Results strongly suggested that GIH negatively regulates the vitellogenin gene expression. Soaking of shrimp nauplii with dsRNA for MIH caused the molt stage advancement of the larvae suggesting that the dsRNA of MIH blocked the expression of MIH and stimulated molting of the larvae. To develop an in vitro culture system for use in the study of gene functions by RNAi in crustaceans, primary cell culture of different tissues was performed. Dissociated cells from the ovary, hepatopancreas, nerve cord and heart of the blue crab Callinectes sapidus were cultured in medium 199. Optimal results were obtained for ovary and hepatopancreas. The culture could be maintained for at least one to two months. Though enhanced green fluorescent protein (EGFP) reporter gene transfection into hepatopancreas primary cells did not achieve a high efficiency, it showed potential as a system to optimize the technique of RNAi and may provide a good starting point for establishing primary crustacean cell lines. In conclusion, these results demonstrated that the RNAi technique can be used to study gene function in M. ensis. The results of the primary cell culture may have potential applications in manipulating growth and reproduction of shrimp and would improve shrimp production in aquaculture. DOI: 10.5353/th_b3689362 Subjects: Double-stranded RNA Gene silencing Neuropeptides Ecdysone Cell culture Shrimps - Genetics


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This dissertation, "Double-stranded RNA Induced Gene Silencing of Neuropeptide Genes in Sand Shrimp, Metapenaeus Ensis and Development of Crustacean Primary Cell Culture" by Haoji, Guan, 關浩基, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation This dissertation, "Double-stranded RNA Induced Gene Silencing of Neuropeptide Genes in Sand Shrimp, Metapenaeus Ensis and Development of Crustacean Primary Cell Culture" by Haoji, Guan, 關浩基, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract of thesis entitled DOUBLE-STRANDED RNA INDUCED GENE SILENCING OF NEUROPEPTIDE GENES IN SAND SHRIMP, METAPENAEUS ENSIS AND DEVELOPMENT OF CRUSTACEAN PRIMARY CELL CULTURE Submitted by Haoji Guan for the degree of Master of Philosophy at The University of Hong Kong in August 2006 The technique using RNA interference (RNAi) to study gene function in different organisms has emerged as a powerful tool in the field of reverse genetics. Although the molecular aspects of RNAi have been studied primarily in insects, there are hints that similar RNAi might occur in the shrimp Metapenaeus ensis as well. The eyestalks of shrimp produce a group of neurohormones that belong to crustacean hyperglycemic hormone (CHH) / molt inhibiting hormone (MIH) / gonad inhibiting hormone (GIH) family. These hormones control diverse processes such as molting and reproduction in M. ensis. MIH is produced in the neurosecretory cells and inhibits molting by suppressing the production of the molting hormone (ecdysone) produced from the Y-organ. GIH plays an inhibitory role in vitellogenesis since GIH mRNA transcript in the eyestalk decreases in the initial phase of gonad maturation and increases towards the end of maturation. In this report, an RNA interference (RNAi) technique was developed to study the function the putative MIH and GIH in the sand shrimp M. ensis. Double stranded RNA for GIH caused the in vitro gene silencing of GIH in the nerve cord in as early as 12 hr. In vivo injection of GIH dsRNA into the female led to the decline of GIH transcript level in the nerve cord, but the vitellogenin (MeVg2) transcript level in the hepatopancreas was increased. Results strongly suggested that GIH negatively regulates the vitellogenin gene expression. Soaking of shrimp nauplii with dsRNA for MIH caused the molt stage advancement of the larvae suggesting that the dsRNA of MIH blocked the expression of MIH and stimulated molting of the larvae. To develop an in vitro culture system for use in the study of gene functions by RNAi in crustaceans, primary cell culture of different tissues was performed. Dissociated cells from the ovary, hepatopancreas, nerve cord and heart of the blue crab Callinectes sapidus were cultured in medium 199. Optimal results were obtained for ovary and hepatopancreas. The culture could be maintained for at least one to two months. Though enhanced green fluorescent protein (EGFP) reporter gene transfection into hepatopancreas primary cells did not achieve a high efficiency, it showed potential as a system to optimize the technique of RNAi and may provide a good starting point for establishing primary crustacean cell lines. In conclusion, these results demonstrated that the RNAi technique can be used to study gene function in M. ensis. The results of the primary cell culture may have potential applications in manipulating growth and reproduction of shrimp and would improve shrimp production in aquaculture. DOI: 10.5353/th_b3689362 Subjects: Double-stranded RNA Gene silencing Neuropeptides Ecdysone Cell culture Shrimps - Genetics

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