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Production of human lysozyme-transgenic cloned porcine embryos by somatic nuclear transfer

Progress in Natural Science:Materials International 2009年第6期19卷 699-704页

作者： Qiuyan Lia,b, Hengxi Wei a, Ying Guoa, Yan Lib, Rui Zhao b, Yufang Maa, Zhengquan Yu a, Bo Tang b, Lei Zhangb, Yunping Dai a,b, Ning Li a, a State Key Laboratory for Agrobiotechnology and Department of Animal Physiology, College of Biological Science, China Agricultural University, No. 2 Yuan-Ming-Yuan West Road, Beijing 100094, China b Ji Pulin Biotech Ltd, Beijing 100094, China State Key Laboratory for Agrobiotechnology and Department of Animal Physiology College of Biological Science China Agricultural University No. 2 Yuan-Ming-Yuan West Road Beijing 100094 China Ji Pulin Biotech Ltd Beijing 100094 China

Due to their physiology and organ size, pigs have significant potential as human disease models and as organ transplantation donors. Genetic modification of pigs could provide benefits for both agriculture and human medicine. In this study, five fetal pig fibroblast cell lines from two species (Wuzhishan and Landrace pigs) were transfected using double-marked human lysozyme (HLY) plasmids (pBC1-HLY-GFP-NEO) by a liposome-mediated method. The ratio of green fluorescent protein (GFP)-expressing cells was >95% in sw7, sw8, slw3 and slw6 cell lines, but only 49.3% in slw9 cells. Cells from the four highly transgenic lines were used as nuclear donors to construct embryos, which were then cultured after fusion and activation by electric stimulation. The rate of cleavage was 76.7%, 48 h after activation. After 7 days, 18.5% of cleaved eggs had developed to the blastocyst stage and 93.3% of blastocysts were GFP-positive. These results indicate that transgenic fetal pig flbroblast cell lines could be obtained by a liposome-mediated method, though the transfection efficiency varied between cell lines. Reconstructed embryos derived from transgenic cells could successfully develop into blastocysts, most of which were GFP-positive.

关键词： EGFP Porcine nuclear transfer Fetal fibroblast Human lysozyme (HLY)

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Experimental cloning of embryos through human-rabbit inter-species nuclear transfer

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Frontiers in Biology 2007年第1期2卷 80-84页

作者： JI Jingjuan GUO Tonghang TONG Xianhong LUO Lihua ZHOU Guixiang FU Yingyun LIU Yusheng Center for Reproductive Medicine Anhui Medical University Affiliated Provincial HospitalHefei 230001China

Therapeutic cloning,which is based on human somatic cell nuclear transfer,is one of our major research *** inter-species nuclear transfer has been introduced to construct human somatic cell cloned embryos,the effects of type,passage,and preparation method of donor cells on embryo development remain *** our experiment,cloned embryos were reconstructed with different passage and preparation methods of ossocartilaginous cell,skin fibroblast,and cumulus *** cumulus cell embryos showed significantly higher development rates than the other two(Pnuclear derivation of the *** result showed that the nuclei of the inter-species cloned embryo cells came from *** conclude that(1)cloned embryos can be constructed through human-rabbit interspecies nuclear transfer;(2)different kinds of somatic cells result in different efficiency of nuclear transfer,while in vitro passage of the donor does not influence embryo development;(3)refrigeration is a convenient and efficient donor cell preparation ***,it is feasible to detect DNA genotype through FISH.

关键词： therapeutic cloning nuclear transfer somatic cell nuclear transfer inter-species cloned embryo

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Factors Affecting the Efficiency of Embryonic Cell nuclear transfer in Rabbits

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Agricultural Sciences in China 2005年第12期4卷 937-942页

作者： CUI Kui-qing LIU Qing-you XIE Ying WEI Jing-wei SHI De-shun Animal Reproduction Institute Guangxi University Nanning 530005 P.R.China

Factors affecting the efficiency of nuclear transfer （NT） in rabbits were examined in the present study. When 100 V mm of pulse strength and 15 us of pulse duration were employed, 3 and 4 electronic pulses resulted in significantly more cytoplasts fused with donor cells compared with 2 electronic pulses （P〈 0.05）, but no significant difference was found in the cleavage rate of reconstructed embryos among the three groups （P〉0.05）. When the duration and number of electronic pulse were fixed at 15 ps and 3 times, increase of pulse intensity from 100 V mm 1 to 150 V mm^-1 and 200 V mm^-1 resulted in a significantly decrease in the cleavage rate of reconstructed embryos （P〈 0.05）, although the fusion rate did not significantly differ among the three groups （P〉 0.05）. Significantly more reconstructed embryos cleaved and developed to blastocysts when they were derived from donor embryos at the 8-16-cell stage, in comparison with the reconstructed embryos derived from donor embryos at the compact morula stage （P 〈 0.05）, although the fusion rate was similar （P 〉 0.05）. Activation of cytoplasts prior to fusion increased the cleavage rate （P〈 0.05） and blastocyst development （P〈 0.05） of reconstructed embryos, but decreased the fusion rate （P 〈 0.05） compared with cytoplasts activated post fusion. More reconstructed embryos developed to blastocysts when they were cultured in TCM ＋ 3% OCS at the first 48 h and then cultured in TCM199＋ 10% FCS, in comparison with the reconstructed embryos cultured in either TCM199＋ 10% FCS or TCM199＋3% OCS （P 〈 0.05）. When 22 NT embryos were transferred into the oviducts of one recipient rabbit, one recipient rabbit delivered a female rabbit at 34 days of gestation. In conclusion, either electrofusion parameter or developmental stage of donor embryos have a significant effect on the efficiency of NT, NT embryos require different concentration of serum at their different development stages.

关键词： Rabbit nuclear transfer Embryo

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Cloning efficiency following ES cell nuclear transfer is influenced by the methylation state of the donor nucleus altered by mutation of DNA methyltransferase 3a and 3b

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Frontiers in Biology 2010年第5期5卷 439-444页

作者： Xiangpeng DAI Xiaoyang ZHAO Hai TANG Jie HAO Jean-Paul RENARD Qi ZHOU Alice JOUNEAU Liu WANG State Key Laboratory of Reproductive Biology Institute of ZoologyChinese Academy of SciencesBeijing 100101China Sino-French Laboratory LABIOCEM Institute of ZoologyChinese Academy of SciencesBeijing 100101China Graduate School of the Chinese Academy of Sciences Beijing 100039China INRA UMR 1198 Biologie du Développement et ReproductionJouy en JosasFrance ENVAMaison-AlfortFrance

The epigenetic state of donor cells plays a vital role in the nuclear reprogramming and chromatin remodel-ing of cloned *** this study we investigated the effect of DNA methylation state of donor cells on the development of mouse embryos reconstructed with embryonic stem(ES)cell *** results conﬁrmed that deletion of the DNA methyltransferase 3a(Dnmt3a)and DNA methyltransferase 3b(Dnmt3b)distinctly decreases the level of DNA methylation in ES *** contrast to wild type ES cells(J1),Dnmt3a–/–3b–/–(DKO)and Dnmt3b–/–(3bKO)donor cells signiﬁcantly elevated the percentage of embryonic stem cell nuclear transfer(ECNT)morula,blastocysts and postimplantation embryos(Pnuclear reprogramming.

关键词： DNA methylation nuclear transfer DNA methyltransferase 3a DNA methyltransferase 3b

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Establishment of customized mouse stem cell lines by sequential nuclear transfer

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Cell Research 2007年第1期17卷 80-87页

作者： Alice Jouneau FRE 2857 Biologie du Développement et Reproduction UMR 1198 ENVA CNRS F-78350 INRA Chinese Academv of Sciences Jouy en Josas Beijing 100080 France State Key Laboratory of Reproductive BiologyJoint laboratory of biology of embryonic cells of mammalians(LABIOCEM) Institute of Zoology China

Therapeutic cloning,whereby embryonic stem cells (ESCs) are derived from nuclear transfer (NT) embryos,mayplay a major role in the new era of regenerative *** this study we established forty nuclear transfer-ESC (NT-ESC) lines that were derived from NT embryos of different donor cell types or *** found that NT-ESCs werecapable of forming embryoid bodies,In addition,NT-ESCs expressed pluripotency stem cell markers in vitro and coulddifferentiate into embryonic tissues in *** embryos from early passage R1 donor cells were able to form full termdeveloped pups,whereas those from late passage R1 ES donor cells lost the potential for reprogramming that is essentialfor live *** subsequently established sequential NT-R1-ESC lines that were developed from NT blastocyst of latepassage R1 ESC ***,these NT-R1-ESC lines,when used as nuclear transfer donors at their early passages,failed to result in live *** indicates that the therapeutic cloning process using sequential NT-ESCs may not rescuethe developmental deficiencies that resided in previous donor generations.

关键词： nuclear transfer therapeutic cloning embryonic stem cells

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nuclear transfer of fish using delayed activating eggs as recipient

Nuclear transfer of fish using delayed activating eggs as re...

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2005水产科技论坛

作者： WU Yan, FU Hong-tuo , GONG Yong-sheng (Freshwater Fisheries Research Center, Key Laboratory of Genetic Breeding of Aquatic Animals and Aquaculture Biology, CAFS Wuxi 214081, China)

＜正＞The effects were assessed using the delayed activating eggs as recipient of nuclear transfer in comparison with normal recipient eggs. After 15 min and 30 min marinating of unfertilized eggs in artificial fertilization experiments, the average hatching rate of fertilized eggs of SOF group was 12. 6% and 5. 6% respectively, but that of control group only 2. 5% and 0. 15%. The fusion rate of SOF group was double that of control group averagely in nuclear transfer experiment, but the hatching rate was similar between them. Artificial fertilization experiments showed Synthetic Ovarian Fluid （SOF） certainly postponed the activation of unfertilized eggs and raised their hatching rate markedly. At the same time, results also indicated the activating state of recipient eggs was not the most important factor of nuclear transfer. Our work is significant theoretically to understand the relationship between activation and development of eggs as well as valuable to improvement of fusion parameters.

关键词： cell fusion nuclear transfer egg activation overmature fish loach

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An Improved Enucleation Method of Bovine Somatic Cell nuclear transfer

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Journal of Genetics and Genomics 2007年第6期34卷 491-496页

作者：华松张志鹏张驰张涌西北农林科技大学生物工程研究所杨凌712100

The aim of this work was to improve the rate of conventionally blind enucleation for bovine somatic cell nuclear transfer. The cross section of a 0.5 ml Eppendorf tube was attached with a sheet of 400 mesh/inch2-cell screen after the bottom of the Eppendorf tube had been cut, and put into a 1 mL Eppendorf tube. In experiment 1, the oocytes in the metaphase Ⅱ stage were placed on the membrane in the Eppendorf tube, and centrifuged at 1,000, 2,000, or 3,000 r/min for 10 min, respectively. The oocytes were stained with Hoechst 33342 and then the relative position of the first polar body to the chromosomes, and the efficiency of enucleation were evaluated. In experiment 2, enucleated oocytes were fused with granulosa cells, following centrifugation and enucleation, and the potential development of the reconstituted embryos was estimated. The results indicated that the rate of enucleation in oocytes after centrifugation at 2,000 r/min for 10 min was 86.6% with an angle less then 20° between the first polar body and chromosomes. The rate of enucleation in cells spun at 2,000 r/min was higher than that of controls （87.4% vs. 64.4%, P 〈 0.05）. Fur- thermore; centrifugation of recipient oocytes did not have a detrimental effect on the development of reconstituted embryos following nuclear transfer. In conclusion, centrifugation assisted enucleation may significantly improve the rate of bovine oocyte enucleation.

关键词： bovine centrifugation nuclear transfer oocyte intraplasmic injection

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Studies on Recipient Rabbit Oocytes in nuclear transfer

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Developmental and Reproductive Biology 1993年第1期2卷 41-46页

作者：邹贤刚肖英达王玉阁杜淼 Institute of Developmental Biology Chinese Academy of Sciences100080 Beijing

Studies were carried out to find out the optimal parameters of enucleation and electroactivation ofrecipient rabbit oocyte for successful nuclear transfer,using the fluorescent stain,DAPI(4-6-diamidino-2-phenylindole),and *** to the position of metaphasechromosomes in relation to the first polar body,the oocytes were classified into three types:*** chromosomes juxtaposed to the polar body;*** with chromosomes in its adjacency; *** with chromosomes further *** relative proportions of each type appeared to varywith the time of maturation at which the oocytes were collected,with those of the later typesincreasing as the maturation process went further *** addition,in-vitro cultivation ofelectroactivated oocytes gave the best results with oocytes that matured in-vito after injection ofovulating hormones(LH or HCG)and oocytes that were cultivated in-vitro for 17-19 *** aresult,it is recommended that oocytes be selected from those collected from the oviducts 13-15 hoursafter injection of LH or HCG,and electrofusion and electroactivation be done aftermicromanipulation and in-vito cultivation for 2-4 *** so doing,it is expected to achieve thehighest enucleation rate of oocytes and the highest fusion rate,the highest activation rate and thehighest development rate of the restructured embryos.

关键词： Rabbit nuclear transfer Oocyte Electroactivation

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Recent Progress of Somatic Cell nuclear transfer in Pigs

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Science Foundation in China 2005年第2期13卷 60-65页

作者： Xu Xiaoming Dou Zhongying Northwest A&F University Shaanxi Branch of National Stern Cell Engineering and Technology Yangling 712100

Research in the field of somatic cell nuclear transfer （SCNT） and transgenic cloning in pigs has become a global hotspot, because porcine organs probably can be the first source of donor organs for human xenotransplantation. In recent years, though great progress has been made in porcine SCNT, the efficiency of nuclear transfer remains very low （〈 1% ）. Thus, it is necessary to improve the procedure of nuclear transfer and to investigate some basic problems further. Recent progress and the related problems of SCNT in pigs are reviewed and analyzed so as to offer some beneficial illumination to researchers.

关键词： nuclear transfer cloning transgene pig

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The Fate of Mitochondria in Ibex-hirus Reconstructed Early Embryos

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Acta Biochimica et Biophysica Sinica 2004年第5期36卷 371-374页

作者： Yan JIANG, Shu-Zhen LIU, Yan-Ling ZHANG, Man-Xi JIANG, Qing-Yuan SUN, and Da-Yuan CHEN* State Key Laboratory of Reproductive Biology, Institute of Zoology, the Chinese Academy of Sciences, Beijing 100080, China State Key Laboratory of Reproductive Biology Institute of Zoology the Chinese Academy of Sciences Beijing 100080 China

Inter-species nuclear transfer could be used to preserve North Goat (Capra ibex), an endan- gered species. We established the culture conditions for ibex-hirus reconstructed embryos and optimized the method for DNA extractions of a single cell and early cloned embryo. By using mitochondria-specific probes of ibex and hirus respectively we found that mitochondria of donor cells can co-exist with recipients in 1-cell and 2-cell stages of the reconstructed embryos but not in the following developmental stages.

关键词： Capra ibex cloned embryos nuclear transfer mitochondria PCR

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