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The integration characteristics of the exogenous growth hormone gene in a transgenic common carp(Cyprinus carpio L.) with fast-growth performance

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Science Bulletin 2015年第19期60卷 1654-1660页

作者： Ji Chen Qing Luo Hairong Bao Lanjie Liao Yongming Li zuoyan zhu Yaping Wang Wei Hu State Key Laboratory of Freshwater Ecology and Biotechnology Institute of Hydrobiology Chinese Academy of Sciences University of Chinese Academy of Sciences

The genetic stability and expression efficiency of exogenous genes in transgenic animals are closely related to integration site and copy number. In our laboratory, by transgenic manipulation and subsequent test crosses, we established an ‘‘all-fish'' growth hormone(GH)transgenic common carp family that exhibits fast *** this present study, genome walking, real-time quantitative polymerase chain reaction, and fluorescence in situ hybridization techniques were applied to identify the integration characteristics of the exogenous grass carp GH gene in the transgenic common carp. The exogenous GH genes, in the form of two complete and one incomplete tandem repeats, were found to have integrated into an ATrich region near the end of a chromosome pair. We hypothesize that the high efficiency of exogenous GH gene expression might be due to the low copy number in the genome and the AT-rich integration site.

关键词： Common carp Growth hormone Transgene Integration site Copy number

Preliminarily study on the maximum handling size, prey size and species selectivity of growth hormone transgenic and non-transgenic common carp Cyprinus carpio when foraging on gastropods

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Journal of Oceanology and Limnology 2018年第4期36卷 1425-1433页

作者： zhu Tingbing ZHANG Lihong ZHANG Tanglin WANG Yaping HU Wei OLSEN Rolf Eric zhu zuoyan State Key Laboratory of Freshwater Ecology and Biotechnology Institute of HydrobiologyChinese Academy of Sciences Wuhan 430072China Yangtze River Fisheries Research lnstitute Chinese Academy of Fisheries Science Wuhan 430223China Department of Biology Norwegian University of Science and Technology7491 TrondheimNorway

The present study preliminarily examined the differences in maximum handling size, prey size and species selectivity of growth hormone transgenic and non-transgenic common carp Cyprinus carpio when foraging on four gastropods species( Bellamya aeruginosa, Radix auricularia, Parafossarulus sinensis and Alocinma longicornis) under laboratory conditions. In the maximum handling size trial, five fish from each age group(1-year-old and 2-year-old) and each genotype(transgenic and non-transgenic) of common carp were individually allowed to feed on B. aeruginosa with wide shell height range. The results showed that maximum handling size increased linearly with fish length, and there was no significant difference in maximum handling size between the two genotypes. In the size selection trial, three pairs of 2-year-old transgenic and non-transgenic carp were individually allowed to feed on three size groups of B. aeruginosa. The results show that the two genotypes of C. carpio favored the small-sized group over the large-sized group. In the species selection trial, three pairs of 2-year-old transgenic and non-transgenic carp were individually allowed to feed on thin-shelled B. aeruginosa and thick-shelled R. auricularia, and five pairs of 2-year-old transgenic and non-transgenic carp were individually allowed to feed on two gastropods species( P. sinensis and A. longicornis) with similar size and shell strength. The results showed that both genotypes preferred thin-shelled Radix auricularia rather than thick-shelled B. aeruginosa, but there were no significant difference in selectivity between the two genotypes when fed on P. sinensis and A. longicornis. The present study indicates that transgenic and non-transgenic C. carpio show similar selectivity of predation on the size-and species-limited gastropods. While this information may be useful for assessing the environmental risk of transgenic carp, it does not necessarily demonstrate that transgenic common carp might have le

关键词： transgenic fish Cyprinus carpio L. fast growth predation ability prey selectivity

Secretogranln-II plays a critical role in zebrafis neurovascular modeling

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Journal of Molecular Cell Biology 2018年第5期10卷 388-401页

作者： Binbin Tao Hongling Hu Kimberly Mitchell Ji Chen Haibo Jia zuoyan zhu Vance L. Trudeau Wei Hu State Key Laboratory of Freshwater Ecology and Biotechnology Institute of Hydrobiology Chinese Academy of Sciences Wuhan 430072 China University of the Chinese Academy of Sciences Beijing 10049 China Department of Biology University of Ottawa Ottawa Ontario K1N 6N5 Canada Key Laboratory of Molecular Biophysics of Ministry of Education College of Life Science and Technology HuaZhong University of Science and Technology Wuhan 430074 China

Secretoneurin （SN） is a neuropeptide derived from specific proteolytic processing of the precursor secretogranin II （Sgll）. In zebrafish and other teleosts, there are two paralogs named sglla and sgllb. Our results showed that neurons expressing sgllb were aligned with central arteries in the hindbrain, demonstrating a close neurovascular association. Both sgllb-/- and sgllo-/-/sgllb-/- mutant embryos were defective in hindbrain central artery development due to impairment of migration and proliferation of central artery cells. Further study revealed that sgllb is non-ceU autonomous and required for central artery development. Hindbrain arterial and venous network identities were not affected in sgllb-/- mutant embryos, and the mRNA levels of Notch and VEGF pathway-related genes were not altered. However, the activation of MAPK and PI3K/AKT pathways was inhibited in sgllb-/- mutant embryos. Reactivation of MAPK or PI3K/AKT in endothelial cells could partially rescue the central artery developmental defects in the sgllb mutants. This studV provides the first in vivo evidence that sgllb ptavs a critical rote in neurovascutar modeling of the hindbrain. Targeting the Sgll system may, therefore, represent a new avenue for the treatment of vascular defects in the central nervous system.

关键词： Secretogranin-II secretoneurin TALENs neurovascular zebrafish

Enzyme-linked immunosorbent assay of changes in serum levels of growth hormone(cGH)in common carps(Cyprinus carpio)

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Science China(Life Sciences) 2008年第2期51卷 157-163页

作者： WU Gang CHEN LiHua ZHONG Shan LI Qi SONG ChaoJun JIN BoQuan zhu zuoyan State Key Laboratory of Freshwater Ecology and Biotechnology Institute of HydrobiologyChinese Academy of SciencesWuhan 430072China Department of Immunology Fourth Military Medical UniversityXi’an 710032China College of Life Science Wuhan UniversityWuhan 430072China

The aim of the present study was to purify the common native carp growth hormone (ncGH), produce monoclonal antibodies (mAbs) to common native carp growth hormone (ncGH), and further enhance the sensitivity of enzyme-linked immunosorbent assays (ELISA) for ncGH. Additionally, we investigated changes in serum ncGH levels in carps raised in different environmental conditions. The recombinant grass carp (Ctenopharyngodon idella) growth hormone was purified and used as antigen to immunize the rabbit. The natural ncGH was isolated from the pituitaries of common carp. SDS-PAGE and Western blot utilizing the polyclonal anti-rgcGH antibody confirmed the purification of ncGH from pituitaries. Purified ncGH was then used as an immunogen in the B lymphocyte hybridoma technique. A total of 14 hybridoma cell lines (FMU-cGH 1-14) were established that were able to stably secrete mAbs against ncGH. Among them, eight clones (FMU-cGH1-6, 12 and 13) were successfully used for Western blot while nine clones (FMU-cGH 1-7, 9 and 10) were used in fluorescent staining and immunohistochemistry. Epitope mapping by competitive ELISA demonstrated that these mAbs recognized five different epitopes. A sensitive sandwich ELISA for detection of ncGH was developed using FMU-cGH12 as the coating mAb and FMU-cGH6 as the enzyme labeled mAb. This detection system was found to be highly stable and sensitive, with detection levels of 70 pg/mL. Additionally, we found that serum ncGH levels in restricted food group and in the net cage group increased 6.9-and 5.8-fold, respectively, when compared to controls, demonstrating differences in the GH stress response in common carp under different living conditions.

关键词： common carp growth hormone monoclonal antibodies ELISA starvation net cage

Acute and chronic un-ionized ammonia toxicity to ‘all-fish’ growth hormone transgenic common carp(Cyprinus carpio L.)

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Chinese Science Bulletin 2010年第35期55卷 4032-4036页

作者： GUAN Bo HU Wei ZHANG TangLin DUAN Ming LI DeLiang WANG YaPing zhu zuoyan State Key Laboratory of Freshwater Ecology and Biotechnology Institute of Hydrobiology Chinese Academy of Sciences Wuhan 430072 China Graduate School of Chinese Academy of Sciences Beijing 100049 China

Ammonia is toxic to fish in natural and artificial *** evaluated the acute(96 h) and chronic(21 d) toxicity of un-ionized ammonia to GH transgenic common carp(Cyprinus carpio L.) and non-transgenic common carp using a static-renewal *** 24,48,72 and 96 h median lethal concentrations(LC50) of un-ionized ammonia were slightly lower in transgenic carp(2.64,2.44,2.28 and 2.16 mg N/L,respectively) than in non-transgenic carp(2.70,2.64,2.52 and 2.33 mg N/L,respectively).Similarly,the median lethal time(LT50) was significantly shorter for transgenic carp(1.41,7.91 and 117.42 h) than for non-transgenic common carp(2.53,14.06 and 150.44 h) following exposure to 3.86,3.29,or 2.09 mg N/L,***,the mortality of transgenic carp was significantly higher than that of non-transgenic carp at all un-ionized ammonia concentrations((0.91 ± 0.12),(0.48 ± 0.06) and(0.12 ± 0.01) mg N/L) during the 21 d chronic toxicity *** results suggest that GH transgenic carp are less tolerant of unionized ammonia than non-transgenic *** data are useful for evaluating potential environmental risk,optimizing stocking density in intensive aquaculture and establishing water quality criteria for ammonia in aquaculture.

关键词：慢性毒性试验生长激素基因非离子氨鲤鱼急性半致死浓度转基因鲤非转基因

Bi-FoRe:an efficient bidirectional knockin strategy to generate pairwise conditional alleles with fluorescent indicators

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Protein & Cell 2021年第1期12卷 39-56页

作者： Bingzhou Han Yage Zhang Xuetong Bi Yang Zhou Christopher JKrueger Xinli Hu zuoyan zhu Xiangjun Tong Bo Zhang Key Laboratory of Cell Proliferation and Differentiation of the Ministry of Education Peking University Genome Editing Research CenterCollege of Life SciencesPeking UniversityBeiing 100871China School of Biology and Biological Engineering South China University of TechnologyGuangzhou 510006China Department of Biomedical Engineering College of EngineeringPeking UniversityBeijing 100871China Wallace H.Coulter Department of Biomedical Engineering Georgia Institute of Techno logy and EmoryAtlantaGA 33032USA Institute of Molecular Medicine Peking UniversityBejing 100871China

Gene expression labeling and conditional manipulation of gene function are important for elaborate dissection of gene ***,contemporary generation of pairwise dual-function knockin alleles to achieve both conditional and geno-tagging effects with a single donor has not been *** we first developed a strategy based on a flipping donor named FoRe to generate conditional knockout alleles coupled with fluorescent allele-labeling through NHEJ-mediated unidirectional targeted insertion in zebrafish facilitated by the CRISPR/Cas *** demonstrated the feasibility of this strategy at sox10 and isl1 loci,and successfully achieved Cre-induced conditional knockout of target gene function and simultaneous switch of the fluorescent reporter,allowing generation of genetic mosaics for lineage *** then improved the donor design enabling efficient one-step bidirectional knockin to generate paired positive and negative conditional alleles,both tagged with two different fluorescent *** introducing Cre recombinase,these alleles could be used to achieve both conditional knockout and conditional gene restoration in parallel;furthermore,differential fluorescent labeling of the positive and negative alleles enables simple,early and efficient realtime discrimination of individual live embryos bearing different genotypes prior to the emergence of morphologically visible *** named our improved donor as Bi-FoRe and demonstrated its feasibility at the sox10 ***,we eliminated the undesirable bacterial backbone in the donor using minicircle DNA *** system could easily be expanded for other applications or to other organisms,and coupling fluorescent labeling of gene expression and conditional manipulation of gene function will provide unique opportunities to fully reveal the power of emerging single-cell sequencing technologies.

关键词： CRISPR/Cas conditional knockout allele labeling conditional rescue minicircle DNA

Surrogate production of genome-edited sperm from a different subfamily by spermatogonial stem cell transplantation

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Science China(Life Sciences) 2022年第5期65卷 969-987页

作者： Fenghua Zhang Yongkang Hao Xianmei Li Yi Li Ding Ye Ru Zhang Xiaosi Wang Mudan He Houpeng Wang zuoyan zhu Yonghua Sun State Key Laboratory of Freshwater Ecology and Biotechnology Hongshan LaboratoryInstitute of HydrobiologyInnovation Academy for Seed DesignChinese Academy of SciencesWuhan 430072China College of Advanced Agricultural Sciences University of Chinese Academy of SciencesBeijing 100049China

The surrogate reproduction technique,such as inter-specific spermatogonial stem cells(SSCs)transplantation(SSCT),provides a powerful tool for production of gametes derived from endangered species or those with desirable ***,generation of genome-edited gametes from a different species or production of gametes from a phylogenetically distant species such as from a different subfamily,by SSCT,has not ***,using two small cyprinid fishes from different subfamilies,Chinese rare minnow(gobiocypris rarus,for brief:Gr)and zebrafish(danio rerio),we successfully obtained Gr-derived genome-edited sperm in zebrafish by an optimized SSCT *** transplanted Gr SSCs supported the host gonadal development and underwent normal spermatogenesis,resulting in a reconstructed fertile testis containing Gr spermatids and zebrafish testicular somatic ***,the surrogate spermatozoa resembled those of host zebrafish but not donor Gr in morphology and swimming *** pou5f3 and chd knockout Gr SSCs were transplanted,Gr-derived genome-edited sperm was successfully produced in *** is the first report demonstrating surrogate production of gametes from a different subfamily by SSCT,and surrogate production of genome-edited gametes from another species as *** method is feasible to be applied to future breeding of commercial fish and livestock.

关键词： surrogate reproduction genome editing spermatogonial stem cell transplantation zebrafish Gobiocypris rarus

Rapid growth and sterility of growth hormone gene transgenic triploid carp

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Chinese Science Bulletin 2011年第16期56卷 1679-1684页

作者： YU Fan XIAO Jun LIANG XiangYang LIU ShaoJun ZHOU GongJian LUO KaiKun LIU Yun HU Wei WANG YaPing zhu zuoyan Key Laboratory of Protein Chemistry and Fish Developmental Biology of Education Ministry of China College of Life Sciences Hunan Normal University Changsha 410081 China State Key Laboratory of Freshwater Ecology and Biotechnology Institute of Hydrobiology Chinese Academy of Sciences Wuhan 430072 China

Triploid carp(100%)with 150(3n=150)chromosomes were obtained by crossing the females of improved tetraploid hybrids(♀, 4n=200)of red crucian carp(♀)×common carp(♂)with the males of diploid yellow river carp(♂,2n=100).The crosses yielded transgenic triploid carp(positive triploid fish,44.2%of the progeny)and non-transgenic triploid carp(negative triploid fish). Histological examination of the gonads of 24-month-old positive triploid fish suggested they were sterile and the fish were not able to produce mature gametes during the breeding ***,both the positive and negative triploid fish were *** had a spindle-shaped,laterally compressed,steel grey body with two pairs of *** of the quantifiable traits of the triploid carp were intermediate between those of the two *** positive and negative triploid fish were raised in the same pond for 2 *** mean body weight of the positive triploid fish was 2.3 times higher than the negative triploid *** weight of the largest positive triploid fish was 2.91 times higher than that of the largest negative triploid ***,we produced fast-growing transgenic triploid carp that have a reduced ecological risk because of their inability to mate and produce progeny.

关键词：三倍体鱼黄河鲤鱼非转基因生长激素基因不育组织学检查横向压缩量化特征

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中国基础研究发展:机遇与挑战

中国科学基金 2005年第4期19卷 193-195页

作者：朱作言国家自然科学基金委员会

值此庆祝中欧建交三十周年之际,中欧双方在北京举办科技战略高层论坛,这是中欧科技交流史上的一件盛事,对筹划未来、推动合作具有深远意义.

关键词：研究发展基础中国科技战略科技交流中欧筹划