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Embryonic and genetic manipulation in fish

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Cell Research 2000年第1期10卷 17-27页

作者： ZHU ZUO YAN YONG HUA SUN (State Key Laboratory of Freshwater ecology and biotechnology, institute Of Hydrobiology, Chinese Academy of Sciences, Luojiashan, Wuhan 430072, China) State Key Laboratory of Freshwater ecology and biotechnology Institute of Hydrobiology Chinese Academy of Sciences Luojiashan China

Fishes, the biggest and most diverse community in vertebrates are good experimental models for studies of cell and developmental biology by many favorable characteristics. Nuclear transplantation in fish has been thoroughly studied in China since 1960s. Fish nuclei of embryonic cells from different genera were transplanted into enucleated eggs generating nucleo-cytoplasmic hybrids of adults. Most importantly, nuclei of cultured goldfish kidney cells had been reprogrammed in enucleated eggs to support embryogenesis and ontogenesis of a fertile fish. This was the first case of cloned fish with somatic cells. Based on the technique of microinjection, recombinant MThGH gene has been transferred into fish eggs and the first batch of transgenic fish were Produced in 1984. The behavior of foreign gene was characterized and the onset of the foreign gene replication occurred between the blastula to gastrula stages and random integration mainly occurred at later stages of embryogenesis. This eventually led to the transgenic mosaicism. The MThGH-transferred common carp enhanced growth rate by 2-4 times in the founder juveniles and doubled the body weight in the adults. The transgenic common carp were more efficient in utilizing dietary protein than the controls. An "all-fish" gene construct CAgcGH has been made by splicing the common carp β-actin gene (CA) promoter onto the grass carp growth hormone gene (goGH) coding sequence. The CAgcGH-transferred Yellow River Carp have also shown significantly fast-growth trait. Combination of techniques of fish cell culture, gene transformation with cultured cells and nuclear transplantation should be able to generate ho- mogeneous strain of valuable transgenic fish to fulfil human requirement in 21st century

关键词： Fish nuclear transplantation transgenic fish gene targeting.

Comparative investigation on spindle behavior and MPF activity changes during oocyte maturation between gynogenetic and amphimictic crucian carp

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Cell Research 1999年第2期9卷 145-154页

作者： YANG ZHONG AN QUN HUA LI YU FENG WANG JIAN FANG GUI (State Key Laboratory of Freshwater ecology and biotechnology, institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China) State Key Laboratory of Freshwater ecology and biotechnology Institute of Hydrobiology Chinese Academy of Sciences Wuhan China

The spindle behavior and MPF activity changes in the progression of oocyte maturation were investigated and compared with cytological observation and kinase assay between gynogenetic silver crucian carp and amphimictic colored crucian carp. MPF activity was measured by using histone H1 as phosphorylation substrate. There were two similar oscillatory MPF kinase activity changes during oocyte maturation in two kinds of fishes with different reproductive modes, but there existed some subtle difference between them. The subtle difference was that the first peak of MPF kinase activity was kept to a longerlasting time in the gynogenetic silver crucian carp than in the amphimictic colored crucian carp. It was suggested that the difference may be related to the spindle behavior changes, such as tripolar spindle formation and spindle rearrangement in the gynogenetic crucian carp.

关键词： Gynogenesis silver crucian carp oocyte maturation MPF kinase tripolar spindle meiosis

Differential screening and characterization analysis of the egg envelope glycoprotein ZP3 cDNAs between gynogenetic and gonochoristic crucian carp

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Cell Research 2001年第1期11卷 17-27页

作者： FAN LIAN CHUN, SHU TING YANG, JIAN FANG GUI (State Key laboratory of Freshwater ecology and biotechnology, institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China) State Key laboratory of Freshwater ecology and biotechnology Institute of Hydrobiology Chinese Academy of Sciences Wuhan 430072 China

Gynogenetic silver crucian carp, Carassius auratus gibelio, is an intriguing model system. In the present work, a systemic study has been initiated by introducing suppression subtractive hybridization technique into this model system to identify the differentially expressed genes in oocytes between gynogenetic silver crucian carp and its closely related gonochoristic color crucian carp. Five differential cDNA fragments were identified from the preliminary screening, and two of them are ZP3 homologues. Moreover, the full length ZP3 cDNAs were cloned from their oocyte cDNA libraries. The length of ZP3 cDNAs were 1378 bp for gyno-carp and 1367 bp for gono-carp, and they can be translated into proteins with 435 amino acids. Obvious differences are not only in the composition of amino acids, but also in the number of potential O-linked oligosaccharide sites. In addition, gyno-carp ZP3 amino acid sequence has an unexpected higher identity value with common carp (83.5%) than that with the closely related gono-carp (74.7%). The unique homology may be originated from the ancient hybridization. Northern blot analysis confirmed that expression of the ZP3 gene occurred exclusively in the oocytes. Because O-linked oligosaccharides on ZP3 have been demonstrated to play very important roles in fertilization, it is suggested that the extra O-linked glycosylation sites may be related to the unique sperm-egg recognition mechanism in gynogenesis.

关键词： Gynogenesis suppression subtractive hybridization glycoprotein ZP3 oocyte maturation.

Quantitative assessment of toxic and nontoxic Microcystis colonies in natural environments using fluorescence in situ hybridization and flow cytometry

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Science China(Life Sciences) 2010年第8期53卷 973-980页

作者： GAN NanQin, HUANG Qun, ZHENG LingLing & SONG LiRong State Key Laboratory of Freshwater ecology and biotechnology, institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China State Key Laboratory of Freshwater ecology and biotechnology Institute of Hydrobiology Chinese Academy of Sciences Wuhan China

Toxic cyanobacterial blooms constitute a threat to human safety because Microcystis sp. releases microcystins during growth, and particularly during cell death. Therefore, analysis of toxic and nontoxic Microcystis in natural communities is required in order to assess and predict bloom dynamics and toxin production by these organisms. In this study, an analysis combining fluorescence in situ hybridization (FISH) with flow cytometry (FCM) was used to discriminate between toxic and nontoxic Microcystis and also to quantify the percentage of toxic Microcystis present in blooms. The results demonstrate that the combination of FISH and flow cytometry is a useful approach for studying the ecology of Microcystis toxin production and for providing an early warning for toxic Microcystis blooms.

关键词： cyanobacteria bloom toxic-microcystis fluorescence in situ hybridization (FISH) flow cytometry (FCM)

Evolutionary mechanisms and practical significance of reproductive success and clonal diversity in unisexual vertebrate polyploids

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Science China(Life Sciences) 2024年第3期67卷 449-459页

作者： Meng Lu Li Zhou Jian-Fang Gui State Key Laboratory of Freshwater ecology and biotechnology Hubei Hongshan Laboratorythe Innovation Academy of Seed DesignInstitute of HydrobiologyChinese Academy of SciencesWuhan 430072China University of Chinese Academy of Sciences Beijing 100049China

Unisexual reproduction is generally relevant to polyploidy, and unisexual vertebrates are often considered an evolutionary “dead end” due to the accumulation of deleterious mutations and absence of genetic diversity. However, some unisexual polyploids have developed strategies to avoid genomic decay, and thus provide ideal models to unveil unexplored evolutionary mechanisms, from the reproductive success to clonal diversity creation. This article reviews the evolutionary mechanisms for overcoming meiotic barrier and generating genetic diversity in unisexual vertebrates, and summarizes recent research advancements in the polyploid Carassius complex. Gynogenetic gibel carp(Carassius gibelio) is a unique amphitriploid that has undergone a recurrent autotriploidy and has overcome the bottleneck of triploid sterility via gynogenesis. Recently, an efficient strategy in which ploidy changes, including from amphitriploid to amphitetraploid, then from amphitetraploid to novel amphitriploid, drive unisexual-sexual-unisexual reproduction transition and clonal diversity has been revealed.Based on this new discovery, multigenomic reconstruction biotechnology has been used to breed a novel strain with superior growth and stronger disease resistance. Moreover, a unique reproduction mode that combines both abilities of ameiotic oogenesis and sperm-egg fusion,termed as ameio-fusiongensis, has been discovered, and it provides an efficient approach to synthesize sterile allopolyploids. In order to avoid ecological risks upon escape and protect the sustainable property rights of the aquaculture seed industry, a controllable fertility biotechnology approach for precise breeding is being developed by integrating sterile allopolyploid synthesis and gene-editing techniques.This review provides novel insights into the origin and evolution of unisexual vertebrates and into the attempts being made to exploit new breeding biotechnologies in aquaculture.

关键词： unisexual vertebrates polyploidy reproduction transition clonal diversity precise breeding genomic reconstruction controllable fertility

Advances in environmental DNA monitoring:standardization,automation,and emerging technologies in aquatic ecosystems

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Science China(Life Sciences) 2024年第7期67卷 1368-1384页

作者： Suxiang Lu Honghui Zeng Fan Xiong Meng Yao Shunping He State Key Laboratory of Freshwater ecology and biotechnology Institute of HydrobiologyChinese Academy of SciencesWuhan 430072China institute of ecology College of Urban and Environmental SciencesPeking UniversityBeijing 100871China School of Life Sciences Peking UniversityBeijing 100871China institute of Deep Sea Science and Engineering Chinese Academy of SciencesSanyaChina Center for Excellence in Animal Evolution and Genetics Chinese Academy of SciencesKunmingChina

Environmental DNA(eDNA)monitoring,a rapidly advancing technique for assessing biodiversity and ecosystem health,offers a noninvasive approach for detecting and quantifying species from various environmental samples.In this review,a comprehensive overview of current eDNA collection and detection technologies is provided,emphasizing the necessity for standardization and automation in aquatic ecological monitoring.Furthermore,the intricacies of water bodies,from streams to the deep sea,and the associated challenges they pose for eDNA capture and analysis are explored.The paper delineates three primary eDNA survey methods,namely,bringing back water,bringing back filters,and bringing back data,each with specific advantages and constraints in terms of labor,transport,and data acquisition.Additionally,innovations in eDNA sampling equipment,including autonomous drones,subsurface samplers,and in-situ filtration devices,and their applications in monitoring diverse taxa are discussed.Moreover,recent advancements in species-specific detection and eDNA metabarcoding are addressed,highlighting the integration of novel techniques such as CRISPR-Cas and nanopore sequencing that enable precise and rapid detection of biodiversity.The implications of environmental RNA and epigenetic modifications are considered for future applications in providing nuanced ecological data.Lastly,the review stresses the critical role of standardization and automation in enhancing data consistency and comparability for robust long-term biomonitoring.We propose that the amalgamation of these technologies represents a paradigm shift in ecological monitoring,aligning with the urgent call for biodiversity conservation and sustainable management of aquatic ecosystems.

关键词： eDNA methodology sampler environment biomonitoring ecogenomic sensors ocean observation instrument

Rational design of a single-component mRNA vaccine against orthopoxvirus and SARS-CoV-2

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Science China(Life Sciences) 2024年第6期67卷 1311-1313页

作者： Yilong Yang Xiaofan Zhao Yu Li Xiaodong Zai Xinyi Wang Yue Zhang Xiaolin Wang Peng Lv Jun Zhang Lihua Hou Junjie Xu Wei Chen Laboratory of Advanced biotechnology Beijing Institute of BiotechnologyAcademy of Military Medical SciencesBeijing 100071China

Dear Editor,In the era of COVID-19,the prevalence of viruses from other families poses new threats to humans.Despite the eradication of smallpox in 1980,the global outbreak of monkeypox in 2022(Lum et al.,2022;Nuzzo et al.,2022)alerted that the re-emerging orthopoxvirus(OPXV)remained a persistent threat to global health.OPXV includes a number of viruses with a broad host range,including variola virus(VARV),monkeypox virus(MPXV),vaccinia virus(VACV).

关键词： al. vaccine Rational

A miniature insertion element transposable in Microcystis sp.FACHB 854

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Progress in Natural Science:Materials International 2006年第5期16卷 486-491页

作者： XU Min, KONG Renqiu and XU Xudong (The State Key Laboratory of Freshwater ecology and biotechnology, institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China) The State Key Laboratory of Freshwater ecology and biotechnology Institute of Hydrobiology Chinese Academy of Sciences Wuhan 430072 China

A 186-bp sequence with imperfect terminal inverted repeats and target direct repeats but without any transposase-encoding capacity was found to be transposable in an isolate derived from Microcystis sp. FACHB 854. This miniature insertion element, designated as ISM854-1 , and with its homologues present at least 10 copies in the genome of Microcystis FACHB 854, is inserted into the 8-bp long and AT-rich target sequences, but none or few in other Microcystis strains. A variant of ISM854-1, denoted ISM854-1A, has perfect inverted repeat sequences and may transpose in pairs in a structure like a composite transposon. This is the first report of non-autonomous transposition of a mini-IS in a cyanobacterium.

关键词： Microcystis miniature IS non-autonomous transposition.

Characterization of transgene integration pattern in F4 hGH-transgenic common carp (Cyprinus carpio L.)

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Cell Research 2005年第6期15卷 447-454页

作者： Bo WU1,*, Yong Hua SUN1,*, Yan Wu WANG1,2, Ya Ping WANG1, Zuo Yan ZHU1,**1 State Key Laboratory of Freshwater ecology and biotechnology, institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China2 College of Life Science, Wuhan University, Wuhan 430072, China State Key Laboratory of Freshwater ecology and biotechnology Institute of Hydrobiology Chinese Academy of Sciences Wuhan China College of Life Science Wuhan University Wuhan China

The integration pattern and adjacent host sequences of the inserted pMThGH-transgene in the F4 hGH-transgeniccommon carp were extensively studied. Here we show that each F4 transgenic fish contained about 200 copies of thepMThGH-transgene and the transgenes were integrated into the host genome generally with concatemers in a head-to-tail arrangement at 4-5 insertion sites. By using a method of plasmid rescue, four hundred copies of transgenes fromtwo individuals of F4 transgenic fish, A and B, were recovered and clarified into 6 classes. All classes of recoveredtransgenes contained either complete or partial pMThGH sequences. The class I, which comprised 83% and 84.5%respectively of the recovered transgene copies from fish A and B, had maintained the original configuration, indicatingthat most transgenes were faithfully inherited during the four generations of reproduction. The other five classes weredifferent from the original configuration in both molecular weight and restriction map, indicating that a few transgeneshad undergone mutation, rearrangement or deletion during integration and germline transmission. In the five types ofaberrant transgenes, three flanking sequences of the host genome were analyzed. These sequences were common carpβ-actin gene, common carp DNA sequences homologous to mouse phosphoglycerate kinase-1 and human epidermalkeratin 14, respectively.

关键词： transgenic common carp plasmid rescue germline transmission integration pattern.