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Association between mitochondrial DNA haplotype compatibility and increased efficiency of bovine intersubspecies cloning

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Journal of Genetics and Genomics 2011年第1期38卷 21-28页

作者： Hao Yan Zhonghai Yan Qingwen Ma Fei Jiao Shuzhen Huang Fanyi zeng yitao zeng Shanghai Institute of Medical Genetics Shanghai Jiao Tong University 24/1400 West Beijing Road Shanghai 200040 China The Key Lab of Embryo Molecular Biology Ministry of Health China and Shanghai Lab of Embryo and Reproduction Engineering 24/1400 West Beijing Road Shanghai 200040 China Institute of Medical Science Shanghai Jiao Tong University School of Medicine 280 South Chongqing Road Shanghai 200025 China

Reconstructed embryos derived from intersubspecies somatic cell nuclear transfer （SCNT） have poorer developmental potential than those from intrasubspecies SCNT. Based on our previous study that Holstein dairy bovine （HD） mitochondrial DNA （mtDNA） haplotype compatibility between donor karyoplast and recipient cytoplast is crucial for SCNT embryo development, we performed intersubspecies SCNT using HD as donor karyoplast and Luxi yellow heifer （LY） as recipient cytoplast according to mtDNA haplotypes determined by polymerase chain reaction- restriction fragment length polymorphism （PCR-RFLP） analysis. The results demonstrated that intersubspecies mtDNA homotype SCNT embryos had higher pre- and post-implantation developmental competence than intrasubspecies mtDNA heterotype embryos as well as improved blastocyst reprogramming status, including normal H3K9 dimethylation pattern and promoter hypomethylation of pluripotent genes such as Oct4 and Sox2, suggesting that intersubspecies SCNT using LY oocytes maintains HD cloning efficiency and may reprogram HD nuclei to develop into a normal cloned animal ultimately. Our results indicated that karyoplast-cytoplast interactions and mtDNA haplotype compatibility may affect bovine intersubspecies SCNT efficiency. This study on bovine intersubspecies SCNT is valuable for understanding the mechanisms of mtDNA haplotype compatibility between karyoplast and cytoplast impacting the bovine SCNT efficiency, and provides an alternative and economic resource for HD cloning.

关键词： Somatic cell nuclear transfer (SCNT) Mitochondria mtDNA haplotype Epigenetic modification Bovine Intersubspecis Intrasubspecis Developmental competence

A Profile of Native Integration Sites Used by φC31 Integrase in the Bovine Genome

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Journal of Genetics and Genomics 2012年第5期39卷 217-224页

作者： Lijuan Qu Qingwen Ma Zaiwei Zhou Haiyan Ma Ying Huang Shuzhen Huang Fanyi zeng yitao zeng Shanghai Institute of Medical Genetics Children＇s Hospital of Shanghai Shanghai Jiao Tong University School of Medicine 24/1400 West Beijing Road Shanghai 200040 China Key Lab of Medical Embryo Molecular Biology Ministry of Health Shanghai 200040 China Shanghai Key Lab of Embryo and Reproduction Engineering Shanghai Municipal Committee of Science and Technology Shanghai 200040 China Institute of Medical Science Shanghai Jiao Tong University School of Medicine 280 South Chongqing Road Shanghai 200025 China

The Streptomyces phage φC31 integrase can efficiently target attB-bearing transgenes to endogenous pseudo attP sites within mammalian genomes. To better understand the activity of φC31 integrase in the bovine genome, DNA sequences of 44 integration events were analyzed, and 32 pseudo attP sites were identified. The majority of these sites share a sequence motif that contains inverted repeats and has similarities to wild-type attP site. Genomic DNA flanking these sites typically contained repetitive sequence elements, such as short and long interspersed repetitive elements. These sequence features indicate that DNA sequence recognition plays an important role in guiding φC31-mediated site-specific integration. In addition, BF27 integration hotspot sites were identified in the bovine genome, which accounted for 13.6% of all isolated integration events and mapped to an intron of the deleted in liver cancer 1 （DLC1） gene. Also we found that the pseudo attP sites in the bovine genome had other features in common with those in the human genome. This study represents the first time that the sequence features of pseudo attP sites specific integrase system has great potential for applied modifications in the bovine genome were analyzed. We conclude that this site- of the bovine genome.

关键词： φC31 integrase Pseudo attP sites Sequence motif Repetitive elements Bovine genome

The Mesenchymal Stem Cells Derived from Transgenic Mice Carrying Human Coagulation Factor Ⅷ Can Correct Phenotype in Hemophilia A Mice

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Journal of Genetics and Genomics 2013年第12期40卷 617-628页

作者： Qing Wang Xiuli Gong Zhijuan Gong Xiaoyie Ren Zhaorui Ren Shuzhen Huang yitao zeng Shanghai Institute of Medical Genetics Children’s Hospital of ShanghaiShanghai Jiao Tong University School of Medicine Key Laboratory of Embryo Molecular Biology Ministry of Health & Shanghai Key Laboratory of Embryo and Reproduction Engineering

Hemophilia A （HA） is an inherited X-linked recessive bleeding disorder caused by coagulant factor VIII （FVIII） deficiency. Previous studies showed that introduction of mesenchymal stem cells （MSCs） modified by FVIll-expressing retrovims may result in phenotypic correction of HA animals. This study aimed at the investigation of an alternative gene therapy strategy that may lead to sustained FVIII transgene expression in HA mice. B-domain-de/eted human FVIll （hFVHIBD） vector was microinjected into single-cell embryos of wild-type mice to generate a transgenic mouse line, from which hFVIIIBD-MSCs were isolated, followed by transplantation into HA mice. RT-PCR and real-time PCR analysis demonstrated the expression of hFVlllBD in multi-organs of recipient HA mice. Immunohistochemistry showed the presence of hFVIIIBD positive staining in multi-organs of recipient HA mice. ELISA indicated that plasma hFVIIIBD level in recipient mice reached its peak （77 ng/ mL） at the 3rd week after implantation, and achieved sustained expression during the 5-week observation period. Plasma FVIII activities of recipient HA mice increased from 0% to 32% after hFVIIIBD-MSCs transplantation. APTT （activated partial thromboplastin time） value decreased in hFVIIIBD-MSCs transplanted HA mice compared with untreated HA mice （45.5 s vs. 91.3 s）. Our study demonstrated an effective phenotypic correction in HA mice using genetically modified MSCs from hFVIIIBD transgenic mice.

关键词： Hemophilia A MSCs FVIII Transgenic mouse Gene therapy

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罕见病的诊断与治疗

上海医学 2022年第8期45卷 515-519页

作者：曾溢滔上海市儿童医院(上海交通大学医学院附属儿童医院) 上海交通大学医学遗传研究所上海200040

罕见病又称为孤儿病,是指患病率极低,但病种繁多、症状严重的一类疾病。罕见病的发病率存在地区和民族间差异,在我国为1/10000。全世界罕见病有7000种,80%属遗传病。50%的罕见病患者在出生时或儿童期发病,30%的罕见病患儿在5岁前病逝,... 详细信息

罕见病又称为孤儿病,是指患病率极低,但病种繁多、症状严重的一类疾病。罕见病的发病率存在地区和民族间差异,在我国为1/10000。全世界罕见病有7000种,80%属遗传病。50%的罕见病患者在出生时或儿童期发病,30%的罕见病患儿在5岁前病逝,故罕见病亟需早诊、早治。罕见病的诊断包括临床实验室检查、家族史调查、染色体基因分析和DNA测序等。罕见病的治疗手段包括调节饮食、替代疗法、孤儿药治疗和基因治疗等,但目前90%的罕见病尚无有效治疗方法。近十年来,国际上针对罕见病的诊断和治疗技术已有很大进步。今后,我国在提高罕见病检测技术水平的同时,应进一步完善三级防治措施,加强罕见病孤儿药的自主研发,推进罕见病的防控和治疗。

关键词：罕见病诊断治疗孤儿药

Expression of biologically active human clotting factor Ⅸ(hFⅨ) in the mammary gland of transgenic mice

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Chinese Science Bulletin 1998年第15期43卷 1294-1298页

作者： HUANG Ying ZHANG Kezhong HUANG Wenying LU Daru HUANG Ying MA Zhanlu REN Zhaorui QIU Xinfang XUE Jinglun zeng yitao HUANG Shuzhen Shanghai Institute of Medical Genetics Shanghai Children’s HospitalShanghai 200040China Institute of Genetics Fudan UniversityShanghai 200433China.

The DNA of human factor Ⅸ (hFⅨ) gene vector pMCⅨm, which had been proven to be able to express in in vitro and living cells, was introduced into 586 zygotes of Kunming White Mice by positive pressure microinjection technique with manual operation. The 499 survival embryos after microinjection were then transferred into pseudopregnant recipient mice and 216 F 0 pups were born. The analysis of PCR and Southern blot hybridization showed that, of the 216, 6 (2 females and 4 males) were integrated with foreign DNA in their genomes, giving an integration frequency of 3% (6/216). Two F\-0 female transgenic mice could express hFⅨ protein in their milk and the content was over 100 ng/mL as measured with ELISA. The biological activities of hFⅨ in the milk of two F\-0 mice were 44 67% and 79 43%, respectively.

关键词： human factorⅨ(hFⅨ) transgenic mice mammary gland expression.

基于咖啡碱和氨基酸中碳氮稳定同位素比率的不同年份普洱生茶真实性认证

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食品工业科技 2023年第5期44卷 1-9页

作者：刘洪林曾艺涛童华荣张凯李照杨天来重庆第二师范学院旅游与服务管理学院重庆400067 重庆市辅仁中学校重庆400067 西南大学食品科学学院重庆400715 重庆市农业技术推广总站重庆401121 重庆市规划和自然资源调查监测院重庆400014

采用咖啡碱和氨基酸中碳氮稳定同位素比率分析方法(气相色谱-燃烧-同位素比值质谱,GC-C-IRMS)开展不同年份普洱生茶真实性认证研究。化学计量学分析表明,基于茶叶咖啡碱和氨基酸中碳氮稳定同位素比率结合化学计量学模型可以对不同年份... 详细信息

采用咖啡碱和氨基酸中碳氮稳定同位素比率分析方法(气相色谱-燃烧-同位素比值质谱,GC-C-IRMS)开展不同年份普洱生茶真实性认证研究。化学计量学分析表明,基于茶叶咖啡碱和氨基酸中碳氮稳定同位素比率结合化学计量学模型可以对不同年份普洱生茶进行有效区分,预测和验证准确率均达到80%以上,其中BP-ANN认证性能最优,预测和验证准确率均达到100%;进一步筛选和鉴定,得到不同年份普洱生茶的3种化学标记,包括δ^(15)N_(Thea)、δ^(13)CG_(lu)和δ^(15)NG_(lu)。因此,基于GC-C-IRMS分析的咖啡碱和氨基酸中碳氮稳定同位素比率分析方法可以用于不同年份普洱生茶的真实性认证。本研究初步建立了普洱生茶产品认证的指标体系及技术体系,也为其他食品的认证研究提供研究思路和方法参考。

关键词：普洱生茶生产年份咖啡碱氨基酸碳氮稳定性同位素比率

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肝细胞有效生成血小板能力的因素分析研究

中华内科杂志 2022年第6期61卷 664-672页

作者：喻梦茹杨冠恒刘光辉曾溢滔薛燕马晴雯曾凡一上海交通大学医学遗传研究所上海市儿童医院上海交通大学医学院附属儿童医院上海200040 上海交通大学基础医学院组织胚胎学与遗传发育学系上海200025 国家卫健委医学胚胎分子生物学重点实验室上海市胚胎与生殖工程重点实验室上海200040

目的探究不同来源的供体造血干细胞(hematopoietic stem cells, HSCs)体内移植后生成血小板(PLT)的差异因素, 进而为解决临床上HSCs移植后PLT重建不良的问题提供新思路。方法通过Ficoll分离技术分别从孕14.5 d(E14.5)胎肝(FL)和8周龄小... 详细信息

目的探究不同来源的供体造血干细胞(hematopoietic stem cells, HSCs)体内移植后生成血小板(PLT)的差异因素, 进而为解决临床上HSCs移植后PLT重建不良的问题提供新思路。方法通过Ficoll分离技术分别从孕14.5 d(E14.5)胎肝(FL)和8周龄小鼠骨髓(BM)中分离HSCs及其下游髓系和淋巴系细胞, 即单个核细胞(MNCs)总和, 移植入清髓辐照后的受体小鼠胫骨骨髓腔内, 构建造血重建FL-MNCs和BM-MNCs小鼠移植治疗模型。定期检测受体小鼠外周血血常规指标。通过流式细胞方法检测供体细胞在受体内的嵌合率及PLT形成上游细胞。进一步利用磁珠分选FL-MNCs和BM-MNCs供体细胞中CD41+巨核细胞, 并诱导PLT生成。另外, 通过定量RT-PCR检测两种来源的CD41+巨核细胞的PLT生成相关基因的表达情况。结果 FL-MNCs移植组和BM-MNCs移植组的血常规参数包括白细胞(WBC)、红细胞(RBC)、PLT均在移植后第4周恢复正常水平, 受体的血细胞基本由供体细胞替代。FL-MNCs移植组比BM-MNCs移植组受体小鼠的PLT水平上升更快, 移植后第4周FL-MNCs组和BM-MNCs组的PLT水平分别为(1.45±0.37)×1012/L和(1.22±0.24)×1012/L, P<0.05。同时, FL-MNCs中含有更高比例的Lin-Sca-1+c-Kit+造血干细胞(FL-MNCs为7.60%±1.40%, BM-MNCs为1.10%±0.46%, P<0.01)、Lin-Sca-1-c-Kit+CD41+CD150+巨核祖细胞(FL-MNCs为3.05%±0.22%, BM-MNCs为0.15%±0.02%, P<0.01), 以及CD41+CD42b+成熟巨核细胞(FL-MNCs为1.60%±0.06%, BM-MNCs为0.87%±0.11%, P<0.01)。体外功能性研究发现胎肝来源的FL-MNCs-CD41+巨核细胞在体外诱导后可更快地生成前血小板样细胞, 第3天即有前血小板样细胞形成, 第5天有明显PLT形成, 而骨髓来源的BM-MNCs-CD41+巨核细胞第5天仍没有前血小板样细胞形成。FL-MNCs-CD41+能表达更高水平的PLT生成相关基因Mpl(3.25倍)、Fog1(3倍)和Gata1(1.5倍)(P<0.05)。结论 FL-MNCs具有更好的受体血小板植入效果以及更快的体外PLT分化速率, 这可能与FL-MNCs细胞组成中较高比例的巨核细胞数量及其Mpl、Fog1和Gata1等相关基因的表达水平有关。这些供体细胞特征的因素分析研究有望为临床治疗造血干细胞移植后PLT重建不良提供供体细胞参考依据。

关键词：肝血小板巨核细胞造血干细胞移植

Expression of human transferrin can be regulated effectively by rabbit transferrin regulatory elements in transgenic mice

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Expression of human transferrin can be regulated effectively...

第八届全国医学生物化学与分子生物学第五届全国临床应用生...

作者： Jingbin Yan Xiuli Gong Shubiao Pan Xinbing Guo Zhaorui Ren yitao zeng Institute of Medical Genetics Children’s Hospital of Shanghai

Human transferrin(hTF) belongs to iron-binding glycoprotein family which plays an important role in iron *** was also a significant factor for the culture of stem ***,the great demand of human transferrin could not be met because it was mainly isolated from the plasma ***,it was not expressed in milks of transgenic animal until now as a result of without proper mammary gland regulatory elements for the expression of hTF,although transgenic bioreactor was a good choice for the production of pharmaceutical ***,it would be useful to improving the expression level of hTF directed by efficient elements through analyzing the its regulatory *** this study,we prepared three expression construction named pcDNA3.1-2 EP-TFN,pcDNA3.1-EP-TFN and pcDNA3.1-RP-TFN,in which the expression of hTF gene was controlled by different rabbit transferring(rTF) regulatory cassette,and the differentiate expression of hTF was investigated in cell lines and transgenic ***,we also analyzed the methylation pattern of rabbit TF promoter in the various tissues of transgenic mice,which might regulate the specific expression of hTF gene in *** results indicated that hTF could be expressed effectively in the serum of transgenic mice when its expression was controlled by rTF promoter and enhancer,the highest expression level reached 913.6 g/***,rTF enhancer would repress the hTF expression in the milks when it was in a series of transgenic mice with rTF *** results also showed that tissue-specific expression of hTF gene was regulated by the methylation status of rTF promoter region,inverse relationship between methylation in rTF promoter and hTF expression level was significantly observed in the livers,hearts and muscles of transgenic *** change of methylation pattern on some transcription factor binding sites would be critical for the regulation of hTF expression in transgenic mice.

关键词： Human transferrin Rabbit transferrin promoter Rabbit transferrin enhancer Methylation pattern Expression

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智能饮品冲调机的设计

包装与食品机械 2022年第2期40卷 43-47页

作者：马新玲曾溢涛牟昊铎林新绅曹霁阳华东理工大学机械与动力工程学院上海200237

针对现有饮品冲调机器难以解决不同物料的自动化加料冲调的问题,设计全自动饮品冲调机——健康先行智能“益喝宝”。通过袋装物料的开袋送料模块、多种物料螺杆送料模块、转轮送料模块、放杯传输模块与冲调搅拌模块的相互配合、协调运行... 详细信息

针对现有饮品冲调机器难以解决不同物料的自动化加料冲调的问题,设计全自动饮品冲调机——健康先行智能“益喝宝”。通过袋装物料的开袋送料模块、多种物料螺杆送料模块、转轮送料模块、放杯传输模块与冲调搅拌模块的相互配合、协调运行,实现饮品物料的全自动冲调。控制系统由人机交互界面HMI接收用户指令,使Arduino控制各模块驱动电机。使用ANSYS进行有限元分析,校核部件力学性能。产品设计新颖实用、市场前景广阔。

关键词：冲调机袋装料包人机交互模块化