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Production of porcine cloned transgenic embryos expressing green fluorescent protein by somatic cell nuclear transfer

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Science china({1.fe Sciences) 2006年第2期49卷 164-171页

作者： ZHANG Yunhai1, PAN Dengke1,2. SUN Xiuzhu1, SUN Guojie1, WANG Xiaobo1, liU Xiaohui1, li Yan1, DAI Yunping1 & li ning1 1. state key laboratory for agrobiotechnology, china agricultural university, beijing 100094, china 2. College of Animal Science and Technology, china agricultural university, beijing 100094, china state {1. {2. for agrobiotechnology China Agricultural University Beijing China College of Animal Science and Technology China Agricultural University Beijing China

In the present study, nuclear transferred embryos (NTEs) were reconstructed by using pig fetal fibroblasts as donors; in vitro matured oocytes as recipients. The effects of G41. selection on donor cells, duration of IVM of prepubertal gilt oocytes; oxygen tension in IVM of oocytes were investigated. The results were as follows: (i) When G41. selected cells expressing GFP were used as donors, the cleavage rate of NTEs decreased drastically in comparison to NTEs derived from donors without antibiotic selection (47.5% vs. 71.6%, p>0.05). For the blastocyst rate, no significant difference was observed between two groups (1.% vs. 1..4%, pduration time was extended from 36 to 42.h (83.6% vs. 96.7%, p>0.05). However, no statistical difference was observed between NTEs derived from oocytes of 36 h IVM group; NTEs from oocytes of 42.h IVM group in the rates of cleavage (59.3% vs. 73.6%, p1..2., p2. tension; NTEs derived from oocytes matured under higher oxygen tension (2.% O2. in cleavage rate (70.6% vs. 67.1., p1..8% vs. 1.2}3%, p1. selection does not have a significant effect on cleavage rate of NTEs expressing GFP. (ii) Nuclear maturation is greatly improved by prolonging IVM duration from 36 to 42.h, while no significant differences were observed for developmental potential of transgenic embryos. Thus IVM 42.h is the better choice in order to obtain maximum number of MII oocytes as recipients. (iii) Lower oxygen tension; higher oxygen tension in IVM have no significant effect on development of cloned embryos.

关键词： transgenic cloning, GFP, Pig, early development.

Expression of Basic Fibroblast Growth Factor Results in the Decrease of Myostatin mRNA in Murine C2.1.0}Myoblasts

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Acta Biochimica et Biophysica Sinica 2006年第10期38卷 697-703页

作者： Hua-Zhong liU~(1,2. Qing li~1 Xing-Yuan YANG~1 lin liU~1 Lei liU~1 Xiao-Rong AN~1* Yong-Fu CHEN~1 1 state key laboratory for agrobiotechnology,College of Biological Science,china agricultural university,{12. 100094,china 2.Modern Biochemistry Center,Guangdong Ocean university,Zhanjiang 52.088,china state {1. {2. for agrobiotechnology College of Biological Science China Agricultural UniversityBeijing 100094 China Modern Biochemistry Center Guangdong Ocean UniversityZhanjiang 524088 China

during the development and regeneration of skeletal muscle,many growth factors,such asbasic fibroblast growth factor (bFGF,FGF-{1. and myostatin,have been shown to play regulating *** contributes to promote proliferation and to inhibit differentiation of skeletal muscle,whereas myostatinplays a series of contrasting *** order to elucidate whether the expression of bFGF has any relationshipwith the expression of myostatin in skeletal muscle cells,we constructed a eukaryotic expression vector forthe expression of exogenous bFGF in murine C{1.1.1.*** RT-PCR assays indicated thatwith the increase of the expression of exogenous bFGF gene,the expression of endogenous myostatin genewas suppressed at mRNA level and protein level.

关键词： basic fibroblast growth factor myostatin murine C2.1.0}myoblast real time RT-PCR

Aberrant histone H4 acetylation in dead somatic cell-cloned calves

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Progress in Natural Science:Materials International 2008年第7期18卷 813-818页

作者： Lei Zhang a,b,*, Shaohua Wang a, Qiang li a, Xiangdong Ding c, Yunping Dai a, ning li a,* a state key laboratory for agrobiotechnology, china agricultural university, beijing 100094, china b beijing Gene & Protein Biotechnology Co. LTD, beijing 100094, china c College of Animal Science and Technology, china agricultural university, beijing 100094, china state {1. {2. for agrobiotechnology China Agricultural University Beijing 100094 China beijing Gene & Protein Biotechnology Co. LTD Beijing 100094 China College of Animal Science and Technology China Agricultural University Beijing 100094 China

In somatic cell-cloned animals, inefficient epigenetic reprogramming can result in an inappropriate gene expression and histone H4 acetylation is one of the key epigenetic modifications regulating gene expression. In this study, we investigated the levels of histone H4 acetylation of 1. development-related genes and expression levels of 1. genes in lungs of three normal control calves and nine aber- rant somatic cell-cloned calves. The results showed that nine studied genes had decreased acetylation levels in aberrant clones (p 0.05). Whereas 1. genes had significantly decreased expression (p 0.05), and only one gene had higher expression level in clones (p 1.genes showed lowered levels of both histone H4 acetylation and expression in aberrant clones than in controls, and the level of histone H4 acetylation was even more lowered in aberrant clones than those in controls. It was suggested that the lower levels of histone H4 acetylation in aberrant clones caused by the previous memory of cell differentiation might not support enough chromatin reprogramming, thus affecting appropriate gene expressions, and growth and development of the cloned calves. To our knowledge, this is the first study on how histone H4 acetylation affects gene expression in organs of somatic cell-cloned calves.

关键词： Somatic cell-cloned calves Histone H4 acetylation Gene expression Lungs

A formal semantics for program debugging

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Science china(Information Sciences) 2012年第1期55卷 133-148页

作者： li Wei1.li {2.,2.1.4} key laboratory of Software Development Environment,School of Computer Science and Engineering,Beihang university,beijing 1.01.1.china 2.8} Ship Research and Development Academy,beijing 1.01.2.china state {1. {2. of Software Development Environment School of Computer Science and Engineering Beihang University Beijing China china Ship Research and Development Academy Beijing China

This work aims to build a semantic framework for automated debugging.A debugging process consists of tracing,locating,and fixing processes *** first two processes are accomplished by a tracing proce{1.re and a locating proce{1.re,*** tracing proce{1.re repro{1.ces the execution of the failed test case with well-designed data structures and saves necessary information for locating *** locating proce{1.re will use the information obtained from the tracing proce{1.re to locate ill-designed statements and to generate a system of fix-equations,whose solution will be used to fix the bugs.A structural operational semantics is given to define the functions of the tracing and locating *** of them are proved to *** main task of fixing process is to solve the *** turns out that for a given failed test case,there exist four types of fix-equations and three dierent solutions:1.The bug is solvable,i.e.,there exists a solution of the system of fix-equations,and the program can be repaired.2.There exists a structural design error in the program,i.e.,the system of fix-equations generated at each round of the locating proce{1.re is solvable,but a new bug will arise when the old bug is being fixed.3)There exists a logical design error,and the system of fix-equations is not solvable.

关键词： debugging operational semantics failed test case fix-equations

Effects of chemical activation and season on birth efficiency of cloned pigs

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Science china({1.fe Sciences) 2009年第7期52卷 657-664页

作者： MA YuFang1,2.li Yan3,WEI HengXi1,li QiuYan3,FANG Rui1,ZHAO Rui3,ZHANG Kun1,XUE Kai1,LOU YanKun1,DAI YunPing3,liAN linSheng2.& li ning1 1 state key laboratory of agrobiotechnology,College of Biological Sciences,china agricultural {12.,beijing 100193,china 2.College of Animal Science and Technology,china agricultural {12.,beijing 100193,china 3 beijing Genprotein Biotechnology Company,beijing 100193,china state {1. {2. of agrobiotechnology College of Biological Sciences China Agricultural University Beijing China College of Animal Science and Technology China Agricultural University Beijing China beijing Genprotein Biotechnology Company Beijing China

The effects of chemical activation on birth efficiency of cloned pigs were studied by investigating the developmental process from porcine oocyte activation to birth of cloned *** different activation methods were used:(i) Electroporation(Ele);(ii) Ele followed by incubation with 6-dimethylaminopurine(6-DMAP);and(iii) Ele followed by a treatment with cycloheximide(CHX).In experiment 1.the rates of cleavage,developmental rates and cell number of porcine parthenogenetic(PA) embryos were investigated in the three treatment *** experiment 2.NT embryos pro{1.ced by the three different activation treatments were compared for the rates of cleavage,development and cell ***,the effects of Ele and Ele+CHX activation methods on birth efficiency of cloned pigs were *** activated oocytes treated by combination activation generally showed a higher(P1.ced more expanded blastocysts than oocytes activated with *** rates of cleavage and total cell number of parthenotes were not significantly *** embryos activated with 6-DMAP developed into blastocyst and expanded blastocyst stages at a significantly(Plity was dramatically decreased in NT *** the CHX activation method,the NT embryo blastocyst rate was substantially(P1.ction of expanded blastocysts was not significantly different from that by the other two *** birth rate of cloned pigs increased in the CHX group,though the rate was not significantly different from *** effects of season on developmental rate of the porcine PA embryos and birth rate of cloned pigs were also examined in our *** oocytes collected in the spring had higher developmental capabilities than those collected in the ***,no difference in birth rate of the cloned pigs was found between the oocytes collected in the two *** re

关键词： parthenogenetic activation chemical activation nuclear transfer pig season

Seasonal influence on the development of parthenotes and cloned embryos in pigs

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Progress in Natural Science:Materials International 2007年第10期17卷 1172-1176页

作者： Zhang Kun1, Zhang Yunhai3,4, Pan Dengke5, Ma Yufang1, Wei Hengxi1, Zhang Jian6, li Xuyang7, Wang Shaohua1, li Yan2. Dai Yunping1,2.and li ning1(1. state key laboratory for agrobiotechnology, College of Biological Science, china agricultural university, beijing 100094, china 2. JiPulin Biotech Ltd., beijing 100094, china 3. Danish Institute of agricultural Sciences, 8830 Tjele, Denmark 4. College of Animal Science and Technology, Anhui agricultural university, Hefei 2.0036, china 5. Institute of Animal Sciences, Chinese Academy of agricultural Sciences, beijing 100094, china 6. Animal Clinical Hospital, china agricultural university, beijing 100094, china 7. Hebei Minghui Group of Pigs Breeding, Sanhe 0652.0, china) state {1. {2. for agrobiotechnology College of Biological Science China Agricultural University Beijing 100094 China Danish Institute of agricultural Sciences 8830 Tjele Denmark College of Animal Science and Technology Anhui Agricultural University Hefei 230036 China Institute of Animal Sciences Chinese Academy of Agricultural Sciences Beijing 100094 China Animal Clinical Hospital China Agricultural University Beijing 100094 China Hebei Minghui Group of Pigs Breeding Sanhe 065200 China JiPulin Biotech Ltd. Beijing 100094 China

Seasonal influence on the developmental ability of porcine embryos was studied by comparisons of the number of cumulus-oocytes-complexes (COCs) per ovary pair and meiotic maturation ability of oocytes in different seasons; and by the observations on developmental competence of the parthenogenetic oocytes and cloned embryos collected from different seasons. We found that the number of COCs per ovary pairs was significantly higher in spring compared to the other seasons (Plined compared to the other seasons (P<0.05), and an increased blastocyst rate of cloned porcine embryos was found in spring compared to autumn and winter (P<0.05). The results suggest that there should be a seasonal influence on the developmental competence of porcine embryos.

关键词： pig cloned parthenogenetic season

A five-fold pig bacterial artificial chromosome library:a resource for positional clo{1. and physical mapping

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Progress in Natural Science:Materials International 2006年第8期16卷 889-892页

作者： liU Wei, ZHANG Ying, liU Zhaoliang, GUO li, WANG Xiaobo, FEI Jing, FENG Jidong, ZHAO Rui, HU Xiaoxiang and li ning (The state key laboratory for Agro-Biotechnology, College of Biology, china agricultural university, beijing 100094, china College of Animal Science and Technology, china agricultural university, beijing 100094, china) The state {1. {2. for Agro-Biotechnology College of Biology China Agricultural University Beijing 100094 China College of Animal Science and Technology China Agricultural University Beijing 100094 China

A pig BAC library was constructed with genomic DNA from a male Erhualian pig. After partial digestion with HindⅢor BamHⅠthe fragments obtained were cloned into the pBeloBAC1. vector. The library consists of 1.432. clones which stored in 480 pieces 384-well plates (2. plates per superpool). A two-step 4-dimension PCR screening system was established to screen the positive clones. An average insert size of 1.3} kb was estimated from 1.5 randomly isolated clones, which indicates that the library is more than five times of genomic coverage. For the demonstration of the probability to pick out any unique genes or DNA markers from the library, 1. single-copy genes were screened out and the positive clones were yielded between 1.and 8 with an average of 3.6. Positive superpools were obtained for 32.microsatellite markers selected from different regions of pig genome. The number of positive superpools for each marker varies from 1.to 9 with an average of 4.78. This BAC library provides an additional resource for pig physical mapping and gene identifica-

关键词： pig BAC library PCR screening system.

Confirmation of a natural hybrid species in Petrocosmea(Gesneriaceae) based on molecular and morphological evidence

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Journal of Systematics and Evolution 2011年第5期49卷 449-463页

作者： 1,3~Zhi-Jing QIU 2.Zu-li YUAN 1~Zhen-Yu li 1~Yin-Zheng WANG 1({2. key laboratory of Systematic and Evolutionary Botany,Institute of Botany,Chinese Academy of Sciences,beijing 100093,china) 2.Henan agricultural university,Zhengzhou 45002.china) 3(Graduate School of the Chinese Academy of Sciences,beijing 100049,china) (state {1. {2. of Systematic and Evolutionary Botany Institute of Botany Chinese Academy of Sciences Beijing 100093 China) (Graduate School of the Chinese Academy of Sciences Beijing 100049 China) (Henan agricultural {1. Zhengzhou 45002 China)

A natural hybrid species in Petrocosmea named Longianthera in Yanshan County,Yunnan Province is confirmed for the first time based on molecular and morphological *** character count procedure of the variable characters show that Longianthera populations are characteristic of the intermediate morphological traits between its putative parents Yanshan and Petrocosmea *** nuclear ribosomal internal transcribed spacer region and three chloroplast regions of matK,trnL-F,and trnT-L are sequenced in the putative hybrid and the related *** a{1.gnment of DNA sequences and the phylogenetic trees could exclude all the other species in Petrocosmea as the parental species except for Yanshan and *** haplotypes in the 31.internal transcribed spacer sequences and six haplotypes in 42.cpDNA sequences were found from 1. individuals of Longianthera *** analyses of DNA sequences,haplotypes,and phylogenetic trees indicate that Longianthera is {1.kely a hybrid species between its putative parents Yanshan and ***,in which Yanshan might be the most possible maternal *** factors may contribute to the natural hybridization between these two parental species in Petrocosmea,such as the overlapped geographic distribution,habitats,flowering periods,and shared ***,the new species of Yanshan and the natural hybrid species of Longianthera are described.

关键词： chloroplast DNA Gesneriaceae ITS morphology natural hybridization Petrocosmea.

Influence of Ordovician carbonate reservoir beds in Tarim Basin by faulting

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Science china Earth Sciences 2008年第S2期51卷 53-60页

作者： L XiuXiang1,2.YANG ning1,2.ZHOU XinYuan3,YANG HaiJun3 & li JianJiao1,2.1 Faculty of Natural Resource & Information Technology,{2. university of Petroleum,beijing 102.49,{2. 2.state key laboratory of Petroleum Resource and Prospecting,{2. university of Petroleum,beijing 102.49,{2. 3 Tarim Oilfield,Petro{2.,Korla 841000,{2. Faculty of Natural Resource & Information Technology China University of Petroleum Beijing China state {1. {2. of Petroleum Resource and Prospecting China University of Petroleum Beijing China Tarim Oilfield PetroChina Korla China

The quality of the Ordovician carbonate reservoir beds in the Tarim Basin is closely related to the development of secondary pores,fractures and cavities. Karstification is important in improving the properties of reservoir beds,and karstification related to unconformity has caught wide attention. Compared with the recent research on the unconformity karst reservoir bed improvement,this paper shows a new way of carbonate reservoir bed transformation. Based on field survey,core and slices observation,transformation of Ordovician carbonate reservoir beds by faulting can be classified into three types: (1. Secondary faults and fracturs generated by faulting improved carbonate reservoir bed properties,which were named the Lunnan or Tazhong82.model; (2. upflow of deep geothermal fluids caused by faulting,with some components metasomatizing with carbonate and forming some secon-dary deposit,such as fluorite. It can improve carbonate reservoir bed properties obviously and is named the Tazhong 82.model; and (3) the faulting extending up to the surface increased the depth of supergene karstification and the thickness of reservoir bed. It is named the Hetianhe model. Trans-formation effect of carbonate reservoir beds by faulting was very significant,mainly distributed on the slopes or on the edge or plunging end of the uplift.

关键词： carbonate faulting reservoir transformation Ordovician Tarim Basin