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夏家辉
《中国工程科学》 2000年 第2卷 第11期 页码 1-11
介绍了中国医学遗传学国家重点实验室在遗传病家系收集、疾病基因定位、疾病基因克隆和疾病基因功能研究方面的研究工作。用细胞遗传学G显带技术于1975年发现了一条与鼻咽癌相关的标记染色体t（1；3）（q44；p11）；1981年将睾丸决定基因（TDF）定位于Yp11.32带；1991年以来收集遗传病家系345种共590个；1996年用显微切割、PCR、微克隆技术克隆了EXT2基因；1998年用基因家族-候选疾病基因克隆方法克隆了遗传性神经性耳聋基因GJB3；1999年用连锁分析和全基因组扫描将一种遗传性弥漫性浅表性光敏性汗孔角化症定位于12q23.2带，并在基因功能研究中发现了一个新的细胞内转运蛋白。
关键词： 遗传病家系 基因定位和克隆 基因家族-候选疾病基因克隆 基因组扫描 基因功能研究
Factors affecting early embryonic development in cattle: relevance for bovine cloning
Yanna DANG, Kun ZHANG
《农业科学与工程前沿（英文）》 2019年 第6卷 第1期 页码 33-41 doi: 10.15302/J-FASE-2018228
Female infertility represents a major challenge for improving the production efficiency in the dairy industry. Historically, fertility has declined whereas milk yield has increased tremendously due to intensive genetic selection. evidence reveals about 60% pregnancy loss takes place during the first month following fertilization. Meanwhile, early embryo development is significant for somatic cell nuclear transfer in cattle as a large proportion of cloned embryos fail to develop beyond peri-implantation stage. Oocyte quality is of utmost importance for the early embryo to develop to term for both fertilized and cloned embryos. Epigenetic reprogramming is a key process occurring after fertilization and critical roles of epigenetic modifiers during preimplantation development are now clear. Incomplete epigenetic reprogramming is believed to be a major limitation to cloning efficiency. Treatment of cloned embryos with epigenetic modifying drugs (e.g., Trichostatin A) could greatly improve cloning efficiency in both mice and cattle. Recently, the rapid progress in high-throughput sequencing technologies has enabled detailed deciphering of the molecular mechanisms underlying these events. The robust efficiency of genomic editing tools also presents an alternative approach to the functional annotation of genes critical to early development.
关键词： bovine cloning embryo development somatic cell nuclear transfer X-inactive specific transcript
柳学周,史宝,王珊珊,徐永江,李晓晓
《中国工程科学》 2014年 第16卷 第9期 页码 50-60
利用RACE技术首次克隆了半滑舌鳎脑垂体中促黄体激素（LH）基因cDNA全长序列。该基因全长670 bp，开放阅读框为477 bp，编码158 个氨基酸。与其他脊椎动物的LH成熟肽氨基酸序列同源性比较表明：半滑舌鳎LH与鲽形目和鲈形目同源性高58 %～68 %。另外，半滑舌鳎LH含有12 个保守的半胱氨酸残基和1 个N-糖基化位点（19～21 NQT）。实时荧光定量组织表达分析表明，LH mRNA在脑、垂体、卵巢等组织中表达，垂体中表达量最丰富，其他组织尤以脑、性腺表达量较高，推测半滑舌鳎LH可能具有广泛的垂体外生理功能；实时荧光定量PCR方法对繁殖周期雌性半滑舌鳎LH表达水平进行测定，结果表明，LH mRNA在Ⅱ～Ⅵ各繁殖周期的脑、垂体、卵巢3 种组织都有表达，但表达水平有差异，在Ⅳ、Ⅴ期表达量最丰富，说明LH主要促进卵母细胞最终成熟及排卵。利用125I 标记的放射免疫测定（RIA）技术检测繁殖周期半滑舌鳎血清LH浓度，结果表明LH在Ⅴ期含量最丰富，也说明LH主要促进卵母细胞最终成熟及排卵。
基于细胞内应变评估的卵母细胞去核方法及其在机器人化克隆中的应用 Article
孙明竹, 刘曜玮, 崔茂盛, 赵启立, 赵相飞, 张艺荻, 黄晶晶, 卢桂章, 赵新
《工程（英文）》 2023年 第24卷 第5期 页码 73-83 doi: 10.1016/j.eng.2022.04.016
自1996 年第一只克隆羊诞生，克隆技术由于在动物育种方面的巨大潜力而得到了很大关注。体细胞核移植技术是动物克隆的核心，其操作过程非常复杂，不可避免地造成细胞内损伤。通常来说，只有不到1%的重构胚胎能够发育成克隆动物，克隆的低成功率是制约其广泛应用的主要原因之一。为此，本文提
出了一种基于细胞内应变评估的卵母细胞去核方法，以减少体细胞核移植中的潜在细胞内损伤，从而提高克隆成功率。首先，根据细胞内速度场计算细胞内应变，进而将内应变作为评价标准来改进去核操作；在此基础上，开发了一套机器人批量体细胞核移植系统，用于实现机器人化克隆。实验结果表明，本文方法将体细胞核移植后的胚胎囊胚率从10.0%提高到20.8%，并成功获得17 头克隆小猪。这是世界上首批由机器人完成克隆操作获得的克隆动物。对比手动核移植操作，本文将克隆成功率从平均0.73%提升到了2.50%，大幅提升了克隆效率。与此同时，基于细胞内应变的去核方法有望应用于其他生物操作，并建立一种通用的减少细胞内损伤的细胞操作规范。
Effects of enucleation method on
Chengcheng ZHAO, Junsong SHI, Rong ZHOU, Ranbiao MAI, Lvhua LUO, Xiaoyan HE, Hongmei JI, Gengyuan CAI, Dewu LIU, Enqin ZHENG, Zhenfang WU, Zicong LI
《农业科学与工程前沿（英文）》 2019年 第6卷 第1期 页码 61-65 doi: 10.15302/J-FASE-2018227
Enucleation is a crucial procedure for mammalian somatic cell nuclear transfer (SCNT), especially for domestic animal cloning. Oocytes of domestic animals such as pigs and cattle contain dark lipid droplets that hinder localization and removal of the nucleus. Using an oocyte enucleation technique that can obtain a high enucleation rate but has minimal negative effects on the reprogramming potential of oocyte for cloning is beneficial for enhancing the outcome of SCNT. In this study, we compared the pig cloning efficiency resulting from blind aspiration-based (BA-B) enucleation and spindle imaging system-assisted (SIS-A) enucleation, and compared the pig SCNT success rate associated with BA-B enucleation and blind aspiration plus post-enucleation staining-based (BAPPS-B) enucleation. SIS-A enucleation achieved a significantly higher oocyte enucleation success rate and tended to obtain a higher full term development rate of SCNT embryos than BA-B enucleation. BAPPS-B enucleation also obtained significantly higher as well as full term development efficiency of cloned porcine embryos than BA-B enucleation. These data indicate that SIS-A and BAPPS-B enucleation are better approaches for pig SCNT than BA-B enucleation.
关键词： cloning enucleation pig SCNT
Embryo-mediated genome editing for accelerated genetic improvement of livestock
Zachariah MCLEAN, Björn OBACK, Götz LAIBLE
《农业科学与工程前沿（英文）》 2020年 第7卷 第2期 页码 148-160 doi: 10.15302/J-FASE-2019305
Selecting beneficial DNA variants is the main goal of animal breeding. However, this process is inherently inefficient because each animal only carries a fraction of all desirable variants. Genome editing technology with its ability to directly introduce beneficial sequence variants offers new opportunities to modernize animal breeding by overcoming this biological limitation and accelerating genetic gains. To realize rapid genetic gain, precise edits need to be introduced into genomically-selected embryos, which minimizes the genetic lag. However, embryo-mediated precision editing by homology-directed repair (HDR) mechanisms is currently an inefficient process that often produces mosaic embryos and greatly limits the numbers of available edited embryos. This review provides a summary of genome editing in bovine embryos and proposes an embryo-mediated accelerated breeding scheme that overcomes the present efficiency limitations of HDR editing in bovine embryos. It integrates embryo-based genomic selection with precise multi-editing and uses embryonic cloning with elite edited blastomeres or embryonic pluripotent stem cells to resolve mosaicism, enable multiplex editing and multiply rare elite genotypes. Such a breeding strategy would enable a more targeted, accelerated approach for livestock improvement that allows stacking of beneficial variants, even including novel traits from outside the breeding population, in the most recent elite genetic background, essentially within a single generation.
关键词： animal breeding cattle cloning CRISPR/Cas9 cytoplasmic injection embryo genome editing germline chimaeras HDR livestock improvement TALENs
Qiong CHEN, Qing YU, Yuhu SONG, Peiyuan Li, Ying CHANG, Zhijun WANG, Lifeng LIU, Wei WU, Jusheng LIN
《医学前沿（英文）》 2009年 第3卷 第2期 页码 148-152 doi: 10.1007/s11684-009-0032-7
关键词： gene X-linked inhibitor of apoptosis protein associated factor-1 (XAF1) promoter transcription regulation
大菱鲆神经肽Y基因克隆及其在工厂化养殖饥饿-投喂策略中的表达特征分析
韩建,刘滨,姜志强,穆小生,雷霁霖
《中国工程科学》 2014年 第16卷 第9期 页码 42-49
本文通过逆转录聚合酶链式反应（RT-PCR）和末端快速扩增技术（RACE）克隆了大菱鲆神经肽Y（NPY）基因全长为729 bp 的互补脱氧核糖核酸（cDNA）序列，其中5′非翻译区（5′-UTR）为70 bp，3′非翻译区（3′-UTR）为359 bp，开放阅读框（ORF）为300 bp。ORF编码含有99 个氨基酸的蛋白质多肽，N端28 个氨基酸为信号肽，信号肽后是由36 个氨基酸组成的NPY成熟肽。针对大菱鲆NPY蛋白质与其他物种的同源性进行比较，结果显示大菱鲆NPY与其他物种的同源性在53.9 %~98 %，特别是与同属于鲆鲽鱼类的牙鲆和美洲拟鲽的同源性高达97 %~98 %，表现出很高的保守性。采用实时定量PCR对大菱鲆NPY基因的表达特征进行分析，结果表明，大菱鲆NPY基因在神经组织和外围组织中都有表达，其中在下丘脑中的表达量最高。饥饿实验中，随着饥饿的进行，NPY的表达量显著变化（P<0.05），呈现先升高再降低，再显著升高并维持一段高水平表达后，再次下降的趋势，饥饿后再投喂会使NPY的表达量升高，并且饥饿时间越长，再投喂后NPY的表达水平越高，但是再投喂2 h 后表达量还会再次下降至相似的水平，这都预示着NPY是一种摄食刺激因子，并且是一种短期的调控机制。
Factors influencing the somatic cell nuclear transfer efficiency in pigs
Yong JIN, Manling ZHANG, Xinrong JU, Shuang LIANG, Qiang XIONG, Lihua ZHAO, Xiaowei NIE, Daorong HOU, Qiang LIU, Junzheng WANG, Chenyu WANG, Xiaokang LI, Lining ZHANG, Xiaorui LIU, Ying WANG, Haiyuan YANG, Yifan DAI, Rongfeng LI
《农业科学与工程前沿（英文）》 2019年 第6卷 第1期 页码 73-80 doi: 10.15302/J-FASE-2018220
Using a data set from our laboratory, we assessed the effects of several factors on pig cloning efficiency. The results demonstrated that cells at high confluence (>90%) used as donor cell resulted in higher pregnancy rate, delivery rate and overall cloning efficiency (number of live offspring born per reconstructed embryo transferred to recipients) compared with the cells at 60% to 79% confluence and 80% to 89% confluence. Cells with four, five and six passages compromised the pregnancy and delivery rates compared with first passage cells. The number of blastocysts transferred by somatic cell nuclear transfer (SCNT) did not significantly affect the cloning efficiency, but transfer of blastocyst derived from culture 5 d after SCNT achieved a significantly higher pregnancy rate compared with one to two cell SCNT embryos from overnight culture. The highest pregnancy rate, delivery rate and the largest litter size were obtained when Bama Miniature pig fibroblasts were used as donor cells and Landrace/Yorkshire hybrid gilts were used as recipients. Recipients treated with chemicals for estrus synchronization had higher pregnancy rates compared with untreated recipients. Our data might be helpful for improving SCNT efficiency in pigs.
关键词： blastocyst donor cell estrus synchronization pregnancy rate pig cloning somatic cell nuclear transfer
标题 作者 时间 类型 操作
Factors affecting early embryonic development in cattle: relevance for bovine cloning
Yanna DANG, Kun ZHANG
期刊论文
Effects of enucleation method on
Chengcheng ZHAO, Junsong SHI, Rong ZHOU, Ranbiao MAI, Lvhua LUO, Xiaoyan HE, Hongmei JI, Gengyuan CAI, Dewu LIU, Enqin ZHENG, Zhenfang WU, Zicong LI
期刊论文
Embryo-mediated genome editing for accelerated genetic improvement of livestock
Zachariah MCLEAN, Björn OBACK, Götz LAIBLE
期刊论文
Cloning of human XAF1 gene promoter and assay of its transcription activity in a variety of cell lines
Qiong CHEN, Qing YU, Yuhu SONG, Peiyuan Li, Ying CHANG, Zhijun WANG, Lifeng LIU, Wei WU, Jusheng LIN
期刊论文
Factors influencing the somatic cell nuclear transfer efficiency in pigs
Yong JIN, Manling ZHANG, Xinrong JU, Shuang LIANG, Qiang XIONG, Lihua ZHAO, Xiaowei NIE, Daorong HOU, Qiang LIU, Junzheng WANG, Chenyu WANG, Xiaokang LI, Lining ZHANG, Xiaorui LIU, Ying WANG, Haiyuan YANG, Yifan DAI, Rongfeng LI
期刊论文
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