Dissertation > Biological Sciences > Molecular Biology > Genetic engineering (genetic engineering)

Construction of zfol Gene Mutant in Zebrafish by Using TALENs

Author Du
Tutor WangHongYing
School Central South University for Nationalities
Course Biochemistry and Molecular Biology
Keywords Lectin Zebrafish Knockout zfol TALENs
Type Master's thesis
Year 2013
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Lectins are widely expressed in animals and plants, with a variety of biologicalactivity. In early embryonic development of fish, the various tissues and organs arenot fully developed, but they still have certain ability to resist pathogens that comefrom the outside world. This resistance is from its nonspecific immunity and maternalimmunity. Lectin existed in the oocyte are considered from maternal immunesubstance and is likely to play an important role in the process of embryonicdevelopent in fish. To gain the information how lectin plays a role in the embryonicdevelopment of fish, a knockout zebrafish line was made to investigate the role oflection.In this study, we cloned zfol gene from zebrafish. The total length of zfol cDNAis937bp containing a ORF with771bp which encodes256amino acids, a5’-UTRwith73bp, and a3’-UTR with93bp. The temporal and spatial expression pattern wasdetermined by using RT-PCR and Western blot analysis. Zfol presents a dynamicexpression pattern in embryonic development.At the beginning, zfol mRNAexpressed with high levels, decreased with development, and increased again. In adultfish, zfol highly expressed in the liver, kidney, ovary, brain, and heart but notdetectable in the testis. Zfol protein was expressed highly in ovary and lower inkidney. Zfol protein was not detected in other tissues. During embryogenesis, the zfolprotein expressed with high levels from0to4hours, decreased in6to9hours, andrised again in12to16hours, decreased in20to24hours, upset at36hours. In orderto determine its role in development, zfol knockout zebrafish were performed byTALENs methods.The zebrafish zfol mutants was made and demonstrated by Western blot analysiswith anti-zfol antibody. The zfol mutants showed that the pericardial cavity hasslightly swollen. The results suggest that zfol may play a role in the regulation ofzebrafish growth and development due to the pericardial cavity is usually caused by streptococcus infection. Therefore, zfol may have a role in protecting againstinfection.To sum up, we clone the zebrafish zfol and determined the expression pattern ofzfol in zebrafish. By using TALENs method, we create the zfol knockout zebrafish andthe mutants showed defects of pericardial cavity.

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