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Prospect of creating trans-genic animals by using sper-matogonial transplantation

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Chinese Science Bulletin 2005年第22期50卷 2537-2545页

作者： bou shorgan The Key Laboratory of National Education Ministry for Mammalian Reproductive Biology and Biotechnology Inner Mongolia University Hohhot 010021 China

Transgenic animal technology is a powerful tool for researching bioscience, biomedicine, bioreactor, and ag- riculture. There are various ways to produce transgenic animals. The most common ways currently available are pronuclear microinjection and nuclear transfer techniques. However, these methods usually result in low efficiency, causing mosaic (in pronuclear microinjection), or develop- mental abnormalities (in nuclear transfer). In 1994, Brinster and his colleagues reported an original method to transfer spermatogonial stem cells from donor to recipient mice. The donor spermatogonia were able to form spermatozoa in re- cipient testes, and to produce progeny carrying the donor’s genetic characters. Since then, a series of novel methods were invented by using spermatogonia transplantation. These new methods facilitate the research and application of sper- matogonia. Some of these methods, when combining with genetic modification methods, will form a novel methodology for creating transgenic animals. The present paper reviews the achievements of research on spermatogonia transplanta- tion related to creating transgenic animal. Such as, trans- plantation techniques, cryopreservation of spermatogonia, preparation of recipients, long-term proliferation of sper- matogonia in culture, genetic modification of spermatogonia, and characterization of germ line transmission of the modi- fied gene, etc. Furthermore the methodologies for creating transgenic animals by using spermatogonia transplantation were described. Based on the difference between donors and recipients used, the methodology is categorized into two groups: allogeneic transplantation, and autologous trans- plantation. Although progress in this research area has been swift, potential difficulties remain to be overcome in each approach. The advantages and existing problems in the methodology are discussed.

关键词：生物科学动物学生物医学显微注射遗传基因

Production of transgenic cashmere goat embryos expressing red fluorescent protein and containing IGF1 hair-follicle-cell specific expression cassette by somatic cell nuclear transfer

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Science China(Life Sciences) 2009年第4期52卷 390-397页

作者： bou shorgan Key Laboratory for Mammalian Reproductive Biology and Biotechnology Ministry of Education Inner Mongolia University

In the present study, cashmere goat fetal fibroblasts were transfected with pCDsR-KI, a hair-follicle-cell specific expression vector for insulin-like growth factor 1 (IGF1) that contains two markers for selection (red fluorescent protein gene and neomycin resistant gene). The transgenic fibroblasts cell lines were obtained after G418 selection. Prior to the somatic cell nuclear transfer (SCNT), the maturation rate of caprine cumulus oocytes complexes (COCs) was optimized to an in vitro maturation time of 18 h. Parthenogenetic ooctyes were used as a model to investigate the effect of two activation methods, one with calcium ionophore IA23187 plus 6-DMAP and the other with ethanol plus 6-DMAP. The cleavage rates after 48 h were respectively 88.7% and 86.4%, with no significant difference (P>0.05). There was no significant difference between the cleavage rate and the blastocyst rate in two different media (SO- Faa and CR1aa; 86.3% vs 83.9%, P>0.05 and 23.1% vs 17.2%,P>0.05). The fusion rate of a 190 V/mm group (62.4%) was significantly higher than 130 V/mm (32.8%) and 200 V/mm (42.9%), groups (P<0.05). After transgenic somatic cell nuclear transfer (TSCNT) manipulation, 203 reconstructed embryos were obtained in which the cleavage rate after in vitro development (IVD) for 48 h was 79.3% (161/203). The blastocyst rate after IVD for 7 to 9 d was 15.3% (31/203). There were 17 embryos out of 31 strongly ex- pressing red fluorescence. Two of the red fluorescent blastocysts were randomly selected to identify transgene by polymerase chain reaction. Both were positive. These results showed that: (i) RFP and Neor genes were correctly expressed indicating that transgenic somatic cell lines and positive trans- genic embryos were obtained; (ii) one more selection at the blastocyst stage was necessary although the donor cells were transgenic positive, because only partially transgenic embryos expressing red fluorescence were obtained; and (iii) through TSCNT manipulation and optimization, transgenic cash- mere goat embryos expr

关键词： cashmere goat insulin like growth factor 1 hair follicle fetal fibroblasts cells transgene somatic cell nuclear transfer

Establishment of Embryonic Stem (ES) Cells Directly Derived From Transgenic Mice Strongly Expressing Greenfluorescence Protein(GFP)

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Establishment of Embryonic Stem (ES) Cells Directly Derived ...

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第一届中华医学会生殖医学分会、中国动物学会生殖生物学分会联合年会

作者： Shorgan BOU Key Laboratory for Mammal Reproduction Biology and Biotechnology of Education Ministry Inner Mongolia University Huhhot 010021 China

GFP-expressing embryonic stem cells are of significance in cell transplantation and usually are established by introducing exogenous EGEP gene into exiting ES cells.This way possibly make genetic c

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Frontiers of Agricultural Science and Engineering 2014年第2期 180-181页

作者： Shorgan BOU Weilun Yin Ning Li Beijing Association for Science and Technology Chinese Society of Forestry State Key Laboratory for Agrobiotechnology in China Agricultural University the Poplar Committee of China Chinese Association of Agricultural Science Societies the Chinese Academy of Engineering(CAE)

Colleagues,It is our great pleasure to introduce you to the Frontiers of Agricultural Science and Engineering(FASE),a new international journal launched in 2014.FASE was founded jointly by the Chinese Academy of Engin...

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Frontiers of Agricultural Science and Engineering 2014年第4期 350-351页

作者： Shorgan BOU Weilun Yin Ning Li Chinese Society of Forestry the Popiar Committee of China Chinese Association of Agricultural Science Societies State Key Laboratory for Agrobiotechnology in China Agricultural University the Chinese Academy of Engineering(CAE) Beijing Association for Science and Technology

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Foreword

Frontiers of Agricultural Science and Engineering 2014年第1期1卷 1-1页

作者： Shorgan BOU Editor-in-chief Vice-presidentChinese Academy of Engineering

In 1994,Lester Brown[1]asked a surprising question which brought global attention to food security:“Who will feed China?”China has no choice but to face this problem.China is producing and will have to produce more food to feed herself.According to the predictions of Koppen’s model[2],in the 21st century,the production of food energy from cultivated land will still be able to meet the qualitative and quantitative demands of the anticipated world population.Obviously,Koppen’s forecasts are optimistic and contrast markedly with Brown’s alarming question.This demonstrates that scientific conclusions need to be fully explored and subjected to continual re-evaluation.

关键词： qualitative Foreword producing

Matrix attachment regions included in a bicistronic vector enhances and stabilizes follistatin gene expressions in both transgenic cells and transgenic mice

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Frontiers of Agricultural Science and Engineering 2016年第1期3卷 87-96页

作者： Xiaoming HU Jing GUO Chunling BAI Zhuying WEI Li GAO Tingmao HU Shorgan BOU Guangpeng LI Key Laboratory of National Education Ministry for Mammalian Reproductive Biology and Biotechnology/Key Laboratory of Herbivore Reproductive Biotechnology and Breeding of Ministry of Agriculture Inner Mongolia University

In the present study, follistatin(FST) gene expression vectors with either a bicistronic gene transfer cassette alone, or a bicistron gene cassette carrying a matrix attachment region(MAR) were constructed and transfected to bovine fetal fibroblasts. Evaluations of both the integration and expression of exogenous FST indicated that the p MAR-CAG-FST-IRES-Ac GFP1-poly A-MAR(pMAR-FST) vector had higher capacity to form monoclonal transgenic cells than the vector without MAR,though transient transfection and integration efficiency were similar with either construct. Remarkably, protein expression in transgenic cells with the p MAR-FST vector was significantly higher than that from the bicistronic vector. Exogenous FST was expressed in all of the p MARFST transgenic mice at F_0, F_1 and F_2. Total muscle growth in F_0 mice was significantly greater than in wild-type mice,with larger muscles in fore and hind limbs of transgenic mice. p MAR-FST transgenic mice were also found with more evenly distributed muscle bundles and thinner spaces between sarcolemma, which suggests a correlation between transgene expression-associated muscle development and the trend of muscle growth. In conclusion, a p MAR-FST vector, which excluded the resistant genes and frame structure, enhances and stabilizes FST gene expressions in both transfected cells and transgenic mice.

关键词： safety of transgenic bicistron gene transfer body transgenic mice muscle development

Attenuation mechanism of Brucella melitensis M5-10,implications for vaccine development and differential diagnosis

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Frontiers of Agricultural Science and Engineering 2014年第4期1卷 331-340页

作者： Yuanqiang ZHENG Yanchun SHI Chang AN Ruisheng LI Dongjun LIU Yuehua KE Kairong MAO Mingjuan YANG Zeliang CHEN Shorgan BOU National Research Center for Animal Transgenic Biotechnology Inner Mongolia UniversityHohhot 010021China Inner Mongolia Key Laboratory of Molecular Biology Inner Mongolia Medical UniversityHohhot 010058China Animal Laboratory Center 302 Hospital of PLABeijing 100039China Institute of Disease Control and Prevention Academy of Military Medical SciencesBeijing 100071China China Institute of Veterinary Drug Control Center for Veterinary Drug EvaluationMinistry of AgricultureBeijing 100081China College of Medicine Shihezi UniversityShihezi 832003China

Brucellosis is a worldwide zoonosis.Vaccination is the most efficient means to prevent and control brucellosis.The current licensed attenuated vaccines for animal use were developed by sequential passage in nonnatural hosts that decreased virulence in its original hosts.The attenuation mechanism of these strains remains largely unknown.In the present study,we sequenced the genome of Brucella melitensis vaccine strain M5-10.Sequence analysis showed that a large number of genetic changes occurred in the vaccine strains.A total of 2854 genetic polymorphic sites,including 2548 SNP,241 INDEL and 65 MNV were identified.Of the 2074 SNPs in coding regions,1310(63.2%)were non-synonymous SNPs.Gene number,percent and N/S ratios were disproportionally distributed among the cog categories.Genetic polymorphic sites were identified in genes of the virB operon,flagella synthesis,and virulence regulating systems.These data indicate that changes in some cog categories and virulence genes might result in the attenuation.These attenuation mechanisms also have implications for screening and development of new vaccine strains.The genetic changes in the genome represent candidate sites for differential diagnosis between these vaccine strains and other virulence ones.Transcription analysis of virulence genes showed that expression of dnaK,vjbR were reduced in M5-10 strain when compared with that in 16M.A duplex PCR targeting virB6 and dnaK was successfully used to differentiate between M5-10 and the virulent 16M strain.The genome re-sequencing technique represents a strong strategy not only for evaluation of vaccines,but also for development of new vaccines.

关键词： Brucella live attenuated vaccine attenuation differential diagnosis

In vivo and in vitro development of Tibetan antelope(Pantholops hodgsonii)interspecific cloned embryos

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Frontiers of Agricultural Science and Engineering 2014年第1期1卷 21-29页

作者： Guanghua SU Lei CHENG Yu GAO Kun LIU Zhuying WEI Chunling BAI Fengxia YIN Li GAO Guangpeng LI Shorgan BOU The Key Laboratory of National Education Ministry for Mammalian Reproductive Biology and Biotechnology Key Laboratory of Herbivore Reproductive Biotechnology and Breeding Ministry of AgricultureInner Mongolia UniversityHohhot 010021China

The Tibetan antelope is endemic to the Tibetan Plateau,China,and is now considered an endangered species.As a possible rescue strategy,the development of embryos constructed by interspecies somatic cell nuclear transfer(iSCNT)was examined.Tibetan antelope fibroblast cells were transferred into enucleated bovine,ovine and caprine oocytes.These cloned embryos were then cultured in vitro or in the oviducts of intermediate animals.Less than 0.5%of the reconstructed antelope-bovine embryos cultured in vitro developed to the blastocyst stage.However,when the cloned antelope-bovine embryos were transferred to caprine oviducts,about 1.6%of the embryos developed to the blastocyst stage.In contrast,only 0.7%of the antelope-ovine embryos developed to the morula stage and none developed to blastocysts in ovine oviducts.The treatment of donor cells and bovine oocytes with trichostatin A did not improve the embryo development even when cultured in the oviducts of ovine and caprine.When the antelope-bovine embryos,constructed from oocytes treated with roscovitine or trichostatin A,were cultured in rabbit oviducts 2.3%and 14.3%developed to blastocysts,respectively.It is concluded that although some success was achieved with the protocols used,interspecies cloning of Tibetan antelope presents difficulties still to be overcome.The mechanisms resulting in the low embryo development need investigation and progress might require a deeper understanding of cellular reprogramming.

关键词： interspecific nuclear transfer bovine ovine caprine oviduct apoptosis