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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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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

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CDH1, a Novel Surface Marker of Spermatogonial Stem Cells in Sheep Testis

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Journal of Integrative Agriculture 2014年第8期13卷 1759-1765页

作者： ZHANG Yan WU Sachula LUO Fen-hua Baiyinbatu LIU Lin-hong HU Tian-yuan YU Bo-yang LI Guang-peng WU Ying-ji The Key Laboratory of National Education Ministry for Mammalian Reproductive Biology and Biotechnology Inner Mongolia University School of Basic Medical Sciences Inner Mongolia Medical University

Spermatogonial stem cells（SSCs） are unique stem cells in adult body that can transmit genetic information to the next generation. They have self-renewal potential and can continuously support spermatogenesis throughout life of a male animal. However, the SSC population is extremely small, isolation and purification of the SSCs is challenging, especially for livestock animals. It has been confirmed that CDH1（cadherin-1, also known as E-cadherin） can be expressed in undifferentiated SSCs of mouse and rats, but it has not been verified in sheep. Here, CDH1 was found as a novel surface marker for sheep SSCs. In this paper, sheep antiCDH1 polyclonal antibodies were prepared and its activity was checked. Using the obtained antibodies and immunohistochemistry analysis, we confirmed that CDH1 can be expressed by SSCs in sheep testis.

关键词： sheep,spermatogonial stem cells,CDH1,polyclonal antibodies,immunofluorescent staining

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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 nuclei.The reprogramming of the somatic nucleus from its original cellular state to a totipotent state is essential for term development after somatic cell nuclear transfer.The nuclear-associated factors contained within oocytes are critical for normal fertilization by sperm or for somatic cell nuclear reprogramming.The chromatin of somatic nuclei can be reprogrammed by factors in the egg cytoplasm whose natural function is to reprogram sperm chromatin.The 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 oocytes.The cytoplasmic milieu most likely contains all of the specific transcription and/or reprogramming factors necessary for cellular reprogramming.Certain transcription factors in the cytoplast may be critical as has been demonstrated for induced pluripotent stem cells.The maternal pronucleus exerts a predominant,transcriptiondependent effect on embryo cytofragmentation,with a lesser effect imposed by the ooplasm and the paternal pronucleus.With 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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Mechanism of C-Phycocyanin Protects Against Low Fertility in Aging Female Mice

Mechanism of C-Phycocyanin Protects Against Low Fertility in...

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中国生理学会生殖科学专业委员会—中国动物学会生殖生物学...

作者： Zhe Han Lei Ge Cheng-Guang Liang The Key Laboratory of National Education Ministry for Mammalian Reproductive Biology and Biotechnology InnerMongolia University

＜正＞Women over 35 years old have higher rates of infertility,largely due to deterioration of oocyte quality.C-Phycocyanin（PC）is a kind of biliprotein enriched in Spirulina platensis,which has been used as anti-oxidant,anti-inflammatory and radical scavenging.In our previous study,we observed a significant increase in litter size after PC administration to D-galactose-induced aging mice.

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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 mammals.Cloned livestock have no apparent health risks,and the quality and safety of the cloned animal products are similar to non-cloned animals.The social behavior and environmental adaptability of postnatal cloned animals,especially when used for grassland farm production purposes,is unknown.In 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 sheep.The main findings of this research were as follows.(1)Under free-grazing conditions,the cloned sheep showed excellent climatic and ecological adaptability.In 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 lambs.Overall,the cloned Dorper sheep exhibited excellent ecological adaptation,which is an important consideration for breeding meat sheep by cloning.The 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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The Dynamic Mechanism of Endogenous Viral MuERV-L/MERVL in Mouse Somatic Cell Nuclear Transfer

The Dynamic Mechanism of Endogenous Viral MuERV-L/MERVL in M...

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2017年第十五届中国北方实验动物科技年会

作者： Lei Yang Lishuang Song Xuefei Liu Lige Bai Guangpeng Li The key laboratory of the national education ministry for mammalian reproductive biology and biotechnology Inner Mongolia University

Despite the success in generating animals through somatic cell nuclear transfer（SCNT） in many different species,a common problem associated with SCNT is the low efficiency.When zygotic（or embryonic）genome activation（ZGA or EGA）,a large number of endogenous retroviruses（ERVs） are expressed,including the murine endogenous retrovirus-L（MuERVL/MERVL）.Here,we generated a series of MERVLreporter mouse stains,which allows us to detect the ZGA of SCNT embryos.We found the majority of SCNT embryos are ZGA failure,and histone H3 lysine 27 trimethylation（H3 K27 me3） as a barrier preventing SCNT reprogramming.Overexpression of the H3 K27 me3 specific demethylases KDM6 A rather than KDM6 B improves the efficiency of SCNT.However,knock-down KDM6 B also facilitates ZGA and consequently improves the SCNT reprogramming.We also revealed that knock-down KDM6 B increased the rate of SCNTderived Duchenne Muscular Dystrophy（DMD） embryonic stem cells establishment.

关键词： SCNT ESCs MERVL embryo KDM6A KDM6B H3K27me3

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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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Establishment and characterization of immortalized bovine male germline stem cell line

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Journal of Integrative Agriculture 2017年第11期16卷 2547-2557页

作者： LEI Qi-jing PAN Qin MA Ju-hong ZHOU Zhe LI Guang-peng CHEN Shu-lin HUA Jin-lian College of Veterinary Medicine Shaanxi Centre of Stem Cells Engineering and TechnologyNorthwest A&F University key laboratory for mammalian reproductive biology and biotechnology Ministry of Education/Inner Mongolia University

Male germline stem cells （mGSCs） are unique adult germ cells with self-renewal potential and spermatogenesis function in the testis. However, further studies are needed to establish a long-term cultural system of mGSCs in vitro, especially for large animals such as bovine mGSCs. In this study, we first established a stable immortalized bovine male germline stem cell line by transducing Simian virus 40 （SV40） large T antigen. The proliferation of these cells was improved significantly. These cells could express spermatogonial stem cell （SSC）-specific markers, such as PLZF, PGP9.5, VASA, LIN28A, and CD49F, both in the mRNA and protein levels. Additionally, these cells could be differentiated into three germ layer cells to enter meiosis, form colonies, and proliferate in the seminiferous tubules of busulfan-induced infertile mice. The immortalized bovine mGSCs maintain the criteria of mGSCs.

关键词： male germline stem cells immortalization bovine proliferation cell transplantation testis

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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 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

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