克隆

①Rat MSC and VSMC were cultured and identified, respectively. MSC were labeled with DAPI firstly, and then co-cultivated with VSMC. The changes of morphology and ultrastructure of co-cultured cells were observed. Immunfluorescence analysis was performed by using monoclonal antibodies against specific antigen.②We established the regulatable system in two steps: a stable MSC line expressing rtTA has been constructed and characterized firstly by transfected with pUHD 17-1hyg and then selected by hygromycin B; in a second step, this line was used for trandfer the AT2R gene to MSC to get the well establishing double stable MSC lines;③The expression of AT2R regulated by doxycycline was evaluated by western blot;④The MSCs were transduced into rat carotid arteries with regulatable AT2R gene after the establishment of rat carotid balloon injury restenosis model. The intimal/medial area ratio were measured by digital analysis system.

研究方法：(1)密度梯度离心法及胶原酶消化法分别培养原代大鼠MSC及VSMC，细胞共培养并行免疫荧光化学染色和透射电镜观察超微结构；(2)组成受Dox调控的哺乳动物表达系统的四种成分的转化、扩增及提纯并酶切鉴定；(3)采用常规分子生物学方法连续两个回合转染体外培养的MSC，并分别采用发光计检测不同细胞克隆萤光素酶活性改变以及RT-PCR方法检测AT2R目的基因mRNA表达情况，根据各个细胞克隆受Dox调控表达的程度，选择低背景、高诱导表达AT2R的细胞系，作为双重稳定MSC细胞系；蛋白免疫印迹法观察该细胞系在Dox调控下AT2R表达的时相性、持续性及在不同浓度Dox调控下的表达情况；(4)建立大鼠颈动脉球囊损伤动物模型，将双重稳定MSC在术中导入血管，分别于14 d、28 d进行病理切片，检测可调控AT2R对新生内膜增生的影响；采用RT-PCR免疫组织化学免疫荧光等技术观察AT2R基因在新生内膜中的表达以及细胞外基质成分表达的改变，TUNEL法检测血管组织中细胞凋亡的变化情况。

Therefore, we selected the 5 genes, as well as SMTN gene related to human cardiovascular diseases, for chromosomal mapping, CDS cloning and analysis, spatio-temporal distribution, mutation riddling, and association analysis with production traits of 3 porcine populations. We expect to know the structure and function of these 6 genes primarily, and supply data for porcine marker assistant selection of improving meat production.

因此，本研究选择NADH呼吸链相关基因，以及与人类心血管疾病相关的SMTN基因，以猪的骨骼肌为研究对象，用人×仓鼠体细胞杂种克隆板进行6个基因的染色体定位、CDS克隆和序列分析、用半定量RT-PCR进行时空表达谱分析、进行突变位点的筛选、并利用3个猪群的产肉性状资料进行基因型与性状的关联分析，以期初步了解这些基因的结构和功能，并为改进猪产肉性能的标记辅助选择提供资料。

HSP90 cDNA fragment cloned from chewing cane(Saccharum officinarum L.) was used as probe to produce a 2 097 bp cDNA fragment by using in silico cloning and splicing.

以克隆到的果蔗 Hsp 90基因片段作为探针，利用电子克隆和序列拼接方法获得一个全长为2 097 bp的cDNA序列。

Desmin, the marker of myoblasts, was adopted to identify the expression of specific marker protein of sarcoblast desmin with immunohistochemistry. Desmin negative cell clones were removed, and desmin cell clones were cultured continuously with the culture fluid replaced once every other day.

采用成肌细胞特异性标志抗原desmin免疫化学染色，鉴定成肌细胞标志蛋白——结蛋白的表达，弃去desmin阴性的细胞克隆，继续培养desmin阳性的细胞克隆，隔天换液1次，7 d进行酶消化传代，获得大量扩增的细胞，并可冻存复苏，用于实验。

To our knowledge, it will be the first gene cloned from M. grisea using the technique of in silico cloning.

这将是第一个用电子克隆技术从稻瘟菌中克隆到的基因。

In silico cloning was developed with the genome and EST project. It is a new method of gene cloning by bioinformatics.

电子克隆是随着基因组计划和EST计划实施而发展起来的利用生物信息学手段进行基因克隆的新方法。

In silico cloning is developing with the project of genomic and EST. It is a new method of gene-cloning through bioinformatics.

电子克隆是随着基因组计划和EST计划实施而发展起来的利用生物信息学手段进行基因克隆的新方法。

In this paper we reviewed the principles of in silico cloning and related bioinformatic resources, and also dicussed the problems and the prospect of in silico cloning.

本文阐述了电子克隆分离杨树功能基因的基本方法、相关的生物信息资源，并讨论了电子克隆技术存在的问题和未来发展。

In silico cloning was a new technique of gene-cloning which developed with genome project and EST project via bioinfomatics method.

电子克隆是随着基因组计划和EST计划的实施而发展起来的，是利用生物信息学手段进行基因克隆的新方法。

In silico cloning was a new strategy of gene cloning developed with the development of genome, EST projects and bio-informatics.

电子克隆是基因克隆的新策略。

Lugalbanda was a god and shepherd king of Uruk where he was worshipped for over a thousand years.

Lugalbanda 是神和被崇拜了一千年多 Uruk古埃及喜克索王朝国王。

I am coming just now,' and went on perfuming himself with Hunut, then he came and sat.

我来只是现在，'歼灭战perfuming自己与胡努特，那麼，他来到和SAT 。