梅花鹿-牛异种体细胞核移植操作方法的研究
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- 英文论文题名：Study on Manipulation of Sika Deer-Bovine Interspecies Somatic Cell Nuclear Transfer
- 论文作者：王士勇 ; 郑军军 ; 杨月春 ; 刘宗岳 ; 于淼 ; 杨福合
- 英文论文作者：Wang Shiyong ; Zheng Junjun ; Yang Yuechun ; Liu Zongyue ; Yu Miao ; Yang Fuhe ; Institute of Specail Animal and Plant Sciences of CAAS ; College of Animal Science ; Jilin Agriculture Science And Technology College
- 年：2016
- 作者机构：中国农业科学院特产研究所;吉林农业科技学院动物科学学院;
- 论文关键词：异种体细胞核移植 ; 盲吸法 ; 挤压法 ; Demecolcine化学辅助去核 ; 带下注核 ; 胞质内注射
- 英文论文关键词：interspecies somatic cell nuclear transfer ; McGrath-Solter method ; extrusion method ; demecoline chemically assisted enucleation ; perivitelline microinjection ; wholecell intracytoplasmic microinjection
- 会议召开时间：2016-06-01
- 会议录名称：“十二五” 鹿科学研究进展
- 语种：中文
- 分类号：Q813
- 学会代码：ZXMY
- 学会名称：中国畜牧业协会
- 页数：8
- 文件大小：490k
- 原文格式：O
摘要
本研究旨在对梅花鹿-牛异种体细胞核移植(Interspecies Somatic Cell Nuclear Transfer,ISCNT)的显微操作方法进行系统研究,从而优化其操作过程。以梅花鹿耳皮肤成纤维细胞为供核细胞,牛MⅡ期卵母细胞为受体,分别采用盲吸法、挤压法和脱羰秋水仙碱(Demecoline,DEME)化学辅助的方法去核,带下注射(Perivitelline Microinjection,PM)、Pizeo破膜细胞胞质内注射(Break-Menbrane-Cell Intracytoplasmic Microinjection,BMCIM)和全细胞胞质内注射(Whole-Cell Intracytoplasmic Microinjection,WCIM)的方法注核,带下注核的卵母细胞电融合后化学激活,其余方法注核后直接化学激活,重组胚用CR1aa培养液体外培养。结果表明:(1)DEME辅助法去核率最高,挤压法最低,三种去核方法的去核率差异显著(P<0.05),去核时间差异不显著(P>0.05);(2)PM组和WCIM组注核成功率分别为100%和94.8%,注核时间分别为31.0s和35.1 s,显著低于BMCIM法(P<0.05),但是注核成功率显著高于BMCIM法(P<0.05),而且两者差异不显著(P>0.05);(3)BMCIM组和WCIM组的激活率均显著高于PM组(P<0.05),其中一步法的BMCIM组最高,一步法和两步法在激活率、卵裂率和囊胚率方面均无显著差异(P>0.05)。结果提示:DEME辅助去核、Pizeo辅助破膜胞质内注射的一步法显微操作可有效用于梅花鹿-牛的异种体细胞核移植的研究。
The purpose of this study was to optimize the manipulation of interspecies somatic cell nuclear transfer(ISCNT) between sika deer and bovine.It was used the sika deer ear skin fibroblast as nuclear donor cell,and bovine M E oocyte as recipient.McGrath-Solter,extrusion and DEME chemically assisted methods were used in enucleation.Perivitelline microinjection(PM),break- membrane- cell intracytoplasmic microinjection(BMCIM)and whole- cell intracytoplasmic microinjection(WCIM) were used in nuclear injection.Oocytes were eleetrofusion after enucleation of PM before chemical activated and others were chemically activated without eleetrofusion.Reconstructed embryos were cultured in media of CRlaa.The results were shown that:(1) The rate of enucleation with the DEME method was the highest,and the rate of extrusion method was the lowest.There was significantly difference(P < 0.05) in the rate of enucleation among all the three enucleate methods,but not significantly difference(P > 0.05) in duration of enucleation.(2) The rate of nuclear injection by PM and WCIM was 100%and 94.8%separately,and the duration of nuclear injection by PM and WCIM was 31.0s and 35.Is separately.There was no difference between them(P> 0.05),their rate of nuclear injection were higher than which in BMCIM group(P <0.05),but duration of nuclear injection was converse(P >0.05).(3) The rate of activation by BMCIM and WCIM group were higher than it in PM group(P < 0.05),and which in BMCIM group of one- step method was the highest.There was no difference among the rate of rate of activation,cleavage and blastocyst between one- step and two- step method(P >0.05).The results suggest that one- step manipulative method of DEME assisted enucleation and pizeo assisted BMCIM was efficient in sika deer- bovine ISCNT.
The purpose of this study was to optimize the manipulation of interspecies somatic cell nuclear transfer(ISCNT) between sika deer and bovine.It was used the sika deer ear skin fibroblast as nuclear donor cell,and bovine M E oocyte as recipient.McGrath-Solter,extrusion and DEME chemically assisted methods were used in enucleation.Perivitelline microinjection(PM),break- membrane- cell intracytoplasmic microinjection(BMCIM)and whole- cell intracytoplasmic microinjection(WCIM) were used in nuclear injection.Oocytes were eleetrofusion after enucleation of PM before chemical activated and others were chemically activated without eleetrofusion.Reconstructed embryos were cultured in media of CRlaa.The results were shown that:(1) The rate of enucleation with the DEME method was the highest,and the rate of extrusion method was the lowest.There was significantly difference(P < 0.05) in the rate of enucleation among all the three enucleate methods,but not significantly difference(P > 0.05) in duration of enucleation.(2) The rate of nuclear injection by PM and WCIM was 100%and 94.8%separately,and the duration of nuclear injection by PM and WCIM was 31.0s and 35.Is separately.There was no difference between them(P> 0.05),their rate of nuclear injection were higher than which in BMCIM group(P <0.05),but duration of nuclear injection was converse(P >0.05).(3) The rate of activation by BMCIM and WCIM group were higher than it in PM group(P < 0.05),and which in BMCIM group of one- step method was the highest.There was no difference among the rate of rate of activation,cleavage and blastocyst between one- step and two- step method(P >0.05).The results suggest that one- step manipulative method of DEME assisted enucleation and pizeo assisted BMCIM was efficient in sika deer- bovine ISCNT.
引文
[1]WILMUT I,SCHNIEKE A E,MCWHIR J,et al.Viable offspring derived from fetal and adult mammalian cells[J].Nature,1997,385(6619):810-813.
[2]KAEDEI Y,FUJIWARA A,TANIHARA F,et al.In Vitro Development Of Cat Interspecies Nuclear Transfer Using Pig's And Cow's Cytoplasm[J].Bull Vet Inst Pulawy,2010,54(3):405-408.
[3]LORTHONGPANICH C,LAOWTAMMATHRON C,CHAN A W,et al.Development of interspecies cloned monkey embryos reconstructed with bovine enucleated oocytes[J].J Reprod Dev,2008,54(5):306-313.
[4]林涛,方南洙,宋君博,等.挤压去核法和盲吸去核法在延边黄牛体细胞核移植中的列比研究[J].江苏农业科学,2009,37(2):178-180.
[5]YIN X J,TANI T,YONEMURA I,et al.Production of cloned pigs from adult somatic cells by chemically assisted removal of maternal chromosomes[J].Biol Reprod,2002,67(2):442-446.
[6]WILLADSEN S M.Nuclear transplantation in sheep embryos[J].Nature,1986,320(6057):63-65.
[7]KIMURA Y,YANAGIMACHI R.Development of normal mice from oocytes injected with secondary spermatocyte nuclei[J].Biol Reprod,1995,53(4):855-862
[8]WAKAYAMA T,PERRY AC,ZUCCOTTI M,et al.Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei[J].Nature,1998,394(6691):369-374.
[9]LEE J W,WU S C,TIAN X C,et al.Production of cloned pigs by whole-cell intracytoplasmic microinjection[J].Biol Reprod,2003,69(3):995-1001.
[10]赵雪萍,张寒莹,王子玉,等.麋鹿耳皮肤成纤维细胞的分离、培养与核型分析[J].南京农业大学学报,2008,31(1):67-71.
[11]杨月春,王士勇,李钟淑,等.延边黄牛体细胞克隆胚胎氧化损伤的初步研究[J].江西农业大学学报,2009,31(6):1069-1073.
[12]董雅娟,柏学进,李建栋,等.牛体细胞核移植技术[J].中国兽医学报,2002,22(4):347-350.
[13]陈自洪,杨素芳,谢英,等.水牛卵母细胞去核方法的比较[J].中国兽医科学,2006,36(6):493-496.
[14]RUSSELL D F,IBANEZ E,ALBERTINI D F,et al.Activated bovine cytoplasts prepared by demecolcine-induced enucleation support development of nuclear transfer embryos in vitro[J].Mol Reprod Dev,2005,72(2):161-170.
[15]COSTA-BORGES N,PARAMIO,MT,CALDERON G,et al.Antimitotic treatments for chemically assisted oocyte enucleation in nuclear transfer procedures[J].Clon Stem Cells,2009,11(1):153-166.
[16]潘晓燕,王正朝,李质馨,等.化学辅助去核法对绵羊卵母细胞去核率和重构胚发育率的影响[J].生物工程学报,2009,25(4):503-508.
[17]SHIN S J,LEE B C,PARK J I,et al.A separate procedure of fusion and activation in an ear fibroblast nuclear transfer program improves preimplantation development of bovine reconstituted oocytes[J].Theriogenology,2001,55(8):1697-1704.
[18]YU Y,DING C,WANG E,et al.Piezo-assisted nuclear transfer affects cloning efficiency and may cause apoptosis[J].Reproduction,2007,133(5):947-954.
[19]CHEN D Y,JIANG M X,ZHAO Z J,et al.Cloning of Asian yellow goat(C.hircus)by somatic cell nuclear transfer;telophase enucleation combined with whole cell intracytoplasmic injection[J].Mol Reprod Dev,2007,74(1):28-34.
[20]LEE B C,KIM M K,JANG G,et al.Dogs cloned from adult somatic cells[J].Nature,2005,436(7051):641.
[21]杨素芳,陈自洪,韦精卫,等.水牛体细胞核移植方法比较及激活前的时间间隔对全细胞胞质内注射法核移植效果的影响[J].畜牧兽医学报,2009,40(12):1735-1740.
[22]ZHOU Q,RENARD J P,LE FRIEC G,et al.Generation of fertile cloned rats by regulating oocyte activation[J].Science,2003,302(5648):1179.
[23]龚国春,戴蕴平,朱化彬,等.供体细胞类型对体细胞克隆牛生产效率的影响[J].中国科学(C辑:生命科学),2004,34(3):257-262.
[2]KAEDEI Y,FUJIWARA A,TANIHARA F,et al.In Vitro Development Of Cat Interspecies Nuclear Transfer Using Pig's And Cow's Cytoplasm[J].Bull Vet Inst Pulawy,2010,54(3):405-408.
[3]LORTHONGPANICH C,LAOWTAMMATHRON C,CHAN A W,et al.Development of interspecies cloned monkey embryos reconstructed with bovine enucleated oocytes[J].J Reprod Dev,2008,54(5):306-313.
[4]林涛,方南洙,宋君博,等.挤压去核法和盲吸去核法在延边黄牛体细胞核移植中的列比研究[J].江苏农业科学,2009,37(2):178-180.
[5]YIN X J,TANI T,YONEMURA I,et al.Production of cloned pigs from adult somatic cells by chemically assisted removal of maternal chromosomes[J].Biol Reprod,2002,67(2):442-446.
[6]WILLADSEN S M.Nuclear transplantation in sheep embryos[J].Nature,1986,320(6057):63-65.
[7]KIMURA Y,YANAGIMACHI R.Development of normal mice from oocytes injected with secondary spermatocyte nuclei[J].Biol Reprod,1995,53(4):855-862
[8]WAKAYAMA T,PERRY AC,ZUCCOTTI M,et al.Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei[J].Nature,1998,394(6691):369-374.
[9]LEE J W,WU S C,TIAN X C,et al.Production of cloned pigs by whole-cell intracytoplasmic microinjection[J].Biol Reprod,2003,69(3):995-1001.
[10]赵雪萍,张寒莹,王子玉,等.麋鹿耳皮肤成纤维细胞的分离、培养与核型分析[J].南京农业大学学报,2008,31(1):67-71.
[11]杨月春,王士勇,李钟淑,等.延边黄牛体细胞克隆胚胎氧化损伤的初步研究[J].江西农业大学学报,2009,31(6):1069-1073.
[12]董雅娟,柏学进,李建栋,等.牛体细胞核移植技术[J].中国兽医学报,2002,22(4):347-350.
[13]陈自洪,杨素芳,谢英,等.水牛卵母细胞去核方法的比较[J].中国兽医科学,2006,36(6):493-496.
[14]RUSSELL D F,IBANEZ E,ALBERTINI D F,et al.Activated bovine cytoplasts prepared by demecolcine-induced enucleation support development of nuclear transfer embryos in vitro[J].Mol Reprod Dev,2005,72(2):161-170.
[15]COSTA-BORGES N,PARAMIO,MT,CALDERON G,et al.Antimitotic treatments for chemically assisted oocyte enucleation in nuclear transfer procedures[J].Clon Stem Cells,2009,11(1):153-166.
[16]潘晓燕,王正朝,李质馨,等.化学辅助去核法对绵羊卵母细胞去核率和重构胚发育率的影响[J].生物工程学报,2009,25(4):503-508.
[17]SHIN S J,LEE B C,PARK J I,et al.A separate procedure of fusion and activation in an ear fibroblast nuclear transfer program improves preimplantation development of bovine reconstituted oocytes[J].Theriogenology,2001,55(8):1697-1704.
[18]YU Y,DING C,WANG E,et al.Piezo-assisted nuclear transfer affects cloning efficiency and may cause apoptosis[J].Reproduction,2007,133(5):947-954.
[19]CHEN D Y,JIANG M X,ZHAO Z J,et al.Cloning of Asian yellow goat(C.hircus)by somatic cell nuclear transfer;telophase enucleation combined with whole cell intracytoplasmic injection[J].Mol Reprod Dev,2007,74(1):28-34.
[20]LEE B C,KIM M K,JANG G,et al.Dogs cloned from adult somatic cells[J].Nature,2005,436(7051):641.
[21]杨素芳,陈自洪,韦精卫,等.水牛体细胞核移植方法比较及激活前的时间间隔对全细胞胞质内注射法核移植效果的影响[J].畜牧兽医学报,2009,40(12):1735-1740.
[22]ZHOU Q,RENARD J P,LE FRIEC G,et al.Generation of fertile cloned rats by regulating oocyte activation[J].Science,2003,302(5648):1179.
[23]龚国春,戴蕴平,朱化彬,等.供体细胞类型对体细胞克隆牛生产效率的影响[J].中国科学(C辑:生命科学),2004,34(3):257-262.