Characterization of zebrafish vitellogenin gene family for potential development of receptor mediated gene transfer method 5

Chapter 5. General conclusions Chapter 5. General conclusions 184 Chapter 5. General conclusions Based on the findings presented in this thesis, the following general conclusions can be drawn: 1) At least seven distinct vitellogenin genes (vtgs) were found in the zebrafish (Danio rerio) genome and they fall into three groups: A (vtg1 and vtg4-7), B (vtg2) and C (vtg3). Vtgs encoded by genes of groups A and C not have the homologous subdomains IV and V; while Vtg2 (group B) has subdomains IV and V. vtg3 was found to encode a Vtg also without the phosvitin domain and may represent a primitive vtg in vertebrate genome. No new vtg sequence was identified from the current zebrafish genome sequence database (86% completion), indicating that there should not be substantially more vtg genes in the zebrafish genome. 2) vtg3 was mapped to chromosome 11 whereas the other five vtgs (vtg1, vtg2, vtg4, vtg5 and vtg7) were mapped to chromosome 22 which likely harbors the vtg6 as well. Phylogenetic analysis and genome mapping indicated that the lineage specific amplification of group A vtgs may occur through tandem duplication of a vtg1-like precursor gene while the vtg precursors leading to the divergence of vtg3 and the other vtg genes might arise from chromosome or whole genome duplication certainly before the radiation of the teleost fish. 3) Expression and quantification studies indicated that in addition to the female liver, vtg mRNAs were also present in several non-liver tissues, including the intestine, ovary, muscle and skin in female fish, and the intestine, testis, muscle, skin and gill in E2 treated male fish. In extra-hepatic tissues examined, the expressions of vtgs were localized mostly in adipocytes. Interestingly, enlarged vascular termini composing mainly of special 185 Chapter 5. General conclusions adipocytes were identified in the ovary and strong vtg expression was detected in these special adipocytes. In the extra-hepatic vtg expressing sites, transcripts of vtg1 and vtg2 were detected; whereas vtg3 mRNAs were barely detectable by in-situ hybridization. 4) In the liver of control female fish, the expression levels of vtg1-3 mRNAs differed, with the strongest expression in vtg1, followed by vtg2 and vtg3, which probably indicates different transcriptional activities of the three vtg genes in zebrafish. Similar trends were also observed in other vtg-expressing tissues. 5) Northern blot analysis showed that the expressions of all seven vtg genes were enhanced in female fish or induced in male fish after E2 treatment. Different folds of increase in the expression levels of vtg1-3 were observed in different tissues of female fish after E2 treatment. In male fish after E2 treatment, the expression levels of vtg1-3 were found to be equal to or even higher than those in corresponding tissues in the control female fish. 6) Red tilapia (Oreochromis mossambica) was used to explore the potential of using Vtgs as DNA carriers for development of a novel method for producing transgenic fish. Preliminary experiments showed that although the purified tilapia Vtg was predominantly taken up by vitellogenic oocytes, the N-succinimidyl 3-(2-pyridyldithio)-propionate (SPDP) modified Vtg moiety in Vtg-polylysine conjugates could not efficiently mediate DNA uptake by oocytes. The reason may be due to degradation or denaturation of Vtgs, destruction of receptor-binding capacity by SPDP modification or improper charge ratio of DNA to Vtg-polylysine in the complexes. 186 Chapter 5. General conclusions 7) As an alternative approach, recombinant Vtg fragments were expressed and shown to have a potential as DNA carriers. To localize the receptor-binding domain in these fragments, seven 6xHis-tagged recombinant tilapia Vtg fragments were expressed in E. coli and four of them were labeled with 32 P and used in the in vivo assay Results indicated that none of these Vtg fragments could be taken up by oocytes specifically, which may be due to the lack of proper posttranslational modification, insufficient zinc, calcium or lipid contents of these Vtg fragments when they were expressed in a procaryotic expression system. In summary, the heterogenicity of vtg gene family and expressions of multiple vtg genes were investigated using a model fish, the zebrafish (Danio rerio). The study of vtg genes provides valuable information for further characterizing the zebrafish vtg family, such as correlating the expression levels or E2 inducibilities of different vtg genes with their promoter elements and/or other trans-acting factors. Results from our systematic investigation of the expression sites of vtg genes challenge the traditional belief that liver is the only organ for Vtg synthesis in oviparous vertebrates. The preliminary studies on receptor-mediated gene transfer using fish Vtgs as DNA carriers encourage future studies, such as on how to improve the construction of conjugates and complexes. 187 . heterogenicity of vtg gene family and expressions of multiple vtg genes were investigated using a model fish, the zebrafish (Danio rerio). The study of vtg genes provides valuable information for further. Red tilapia (Oreochromis mossambica) was used to explore the potential of using Vtgs as DNA carriers for development of a novel method for producing transgenic fish. Preliminary experiments showed. Chapter 5. General conclusions Chapter 5. General conclusions 184 Chapter 5. General conclusions Based on the findings presented in this thesis, the following general conclusions