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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 optim

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

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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

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Oocyte-associated transcription factors in reprogramming after somatic cell nuclear transfer:a review

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Frontiers of Agricultural Science and Engineering 2014年第2期1卷 104-113页

作者： Fengxia YIN Hui LIU shorgan bou Guangpeng LI The Key Laboratory of National Education Ministry for Mammalian Reproductive Biology and Biotechnology the College of Life SciencesInner Mongolia UniversityHohhot 010070China

Oocytes are unique cells with the inherent capability to reprogram *** reprogramming of the somatic nucleus from its original cellular state to a totipotent state is essential for term development after somatic cell nuclear *** nuclear-associated factors contained within oocytes are critical for normal fertilization by sperm or for somatic cell nuclear *** chromatin of somatic nuclei can be reprogrammed by factors in the egg cytoplasm whose natural function is to reprogram sperm *** oocyte first obtains its reprogramming capability in the early fetal follicle,and then its capacity is enriched in the late growth phase and reaches its highest capability for reprogramming as fully-grown germinal vesicle *** cytoplasmic milieu most likely contains all of the specific transcription and/or reprogramming factors necessary for cellular *** transcription factors in the cytoplast may be critical as has been demonstrated for induced pluripotent stem *** maternal pronucleus exerts a predominant,transcriptiondependent effect on embryo cytofragmentation,with a lesser effect imposed by the ooplasm and the paternal *** deep analysis of transcriptomics in oocytes and early developmental stage embryos more maternal transcription factors inducing cellular reprogramming will be identified.

关键词： nuclear reprogramming somatic cell transcription factors transcriptomics

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

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 *** way possibly make genetic characterization of ES cells to be changed,which further affect cell differentiation in vitro or *** we report the establishment of GFP-expressing embryonic stem cells directly derived from inner cell mass (ICM)of blastocysts of EGFP-transgenic *** results show resultant ES cells express high levels of green fluorescence protein(GFP),maintain the developmental potential to form teratoma with all three embryonic germ *** ES cells are immunoreacfive for alkaline phosphatase ***-4、 *** type of ES cells can be largely applied in cell differentiation and transplantation by tracing the EGF protein.

关键词： EGFP-transgenic mouse Embryonic stem cells Green fluorescence protein

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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

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Invitation to Contribute to FASE

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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 the Chinese Academy of Engineering(CAE) Chinese Society of Forestry Chinese Association of Agricultural Science Societies the Poplar Committee of China State Key Laboratory for Agrobiotechnology in China Agricultural University Beijing Association for Science and Technology

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

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Invitation to Contribute to FASE

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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 the Chinese Academy of Engineering(CAE) Chinese Society of Forestry Chinese Association of Agricultural Science Societies the Popiar Committee of China State Key Laboratory for Agrobiotechnology in China Agricultural University Beijing Association for Science and Technology

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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 *** is the most efficient means to prevent and control *** current licensed attenuated vaccines for animal use were developed by sequential passage in nonnatural hosts that decreased virulence in its original *** attenuation mechanism of these strains remains largely *** the present study,we sequenced the genome of Brucella melitensis vaccine strain *** 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 *** the 2074 SNPs in coding regions,1310(63.2%)were non-synonymous *** number,percent and N/S ratios were disproportionally distributed among the cog *** polymorphic sites were identified in genes of the virB operon,flagella synthesis,and virulence regulating *** data indicate that changes in some cog categories and virulence genes might result in the *** attenuation mechanisms also have implications for screening and development of new vaccine *** genetic changes in the genome represent candidate sites for differential diagnosis between these vaccine strains and other virulence *** 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 *** 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

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The ecological adaptability of cloned sheep to free-grazing in the Tengger Desert of Inner Mongolia,China

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Frontiers of Agricultural Science and Engineering 2014年第3期1卷 191-200页

作者： Xinxin LI Huijuan WANG Guanghua SU Zhuying WEI Chunling BAI Wuni-MENGHE Yanhui HOU Changqing YU shorgan bou Guangpeng LI The Key Laboratory of National Education Ministry for Mammalian Reproductive Biology and Biotechnology Key Laboratory of Herbivore Reproductive Biotechnology and Breeding of Ministry of AgricultureInner Mongolia UniversityHohhot 010070China The Inner Mongolia Rangeland Ecology Institute Alashan 750306China

Since the birth of the first cloned sheep,somatic cell nuclear transfer technology has been successfully used to clone a variety of *** livestock have no apparent health risks,and the quality and safety of the cloned animal products are similar to non-cloned *** social behavior and environmental adaptability of postnatal cloned animals,especially when used for grassland farm production purposes,is *** the present study,the cloned Dorper sheep equipped with GPS location devices were free-grazed in a harsh natural environment similar to conditions commonly experienced by Mongolian *** main findings of this research were as follows.(1)Under free-grazing conditions,the cloned sheep showed excellent climatic and ecological *** extreme temperature conditions ranging from–30 to 40°C,the cloned sheep maintained acceptable body condition and behaved as other sheep.(2)The cloned sheep quickly adapted from a herd feeding strategy to the harsh environment and quickly exhibited a grazing regimen as other free-grazing sheep.(3)The cloned sheep exhibited free-grazing patterns and social behavior as other sheep.(4)The cloned sheep in the harsh environment thrived and produced healthy ***,the cloned Dorper sheep exhibited excellent ecological adaptation,which is an important consideration for breeding meat sheep by *** Dorper sheep readily adapted to the free-grazing conditions on the Mongolian plateau grassland,which attests to their ability to withstand harsh environmental conditions.

关键词： somatic cell nuclear transfer free-grazing synchronization Dorper sheep cloned animal ecology

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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 *** a possible rescue strategy,the development of embryos constructed by interspecies somatic cell nuclear transfer(iSCNT)was *** antelope fibroblast cells were transferred into enucleated bovine,ovine and caprine *** cloned embryos were then cultured in vitro or in the oviducts of intermediate *** than 0.5%of the reconstructed antelope-bovine embryos cultured in vitro developed to the blastocyst ***,when the cloned antelope-bovine embryos were transferred to caprine oviducts,about 1.6%of the embryos developed to the blastocyst *** contrast,only 0.7%of the antelope-ovine embryos developed to the morula stage and none developed to blastocysts in ovine *** 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 *** 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,*** is concluded that although some success was achieved with the protocols used,interspecies cloning of Tibetan antelope presents difficulties still to be *** 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

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