微细胞介导人14号染色体自A9细胞转移至DT40细胞

为了获得含人14号染色体的DT40细胞,用于人抗体基因的表达研究.本研究利用微细胞介导的染色体转移技术,将A9细胞中的人14号染色体转移至DT40细胞中.首先,摸索秋水仙胺诱导A9细胞微核形成最佳浓度与最佳时间,以终浓度为10 mg/mL的细胞松驰素B破坏细胞骨架,离心分离微细胞,获得的微细胞依次经8μm、5μm、3μm滤膜过滤后与受体细胞DT40融合,细胞铺板后加入G418筛选.然后,对长出的抗性克隆进行基因组DNA检测及FISH杂交,分析人14号染色体在DT40杂合细胞克隆中的存在情况.结果显示,成功获得含人14号染色体的DT40(#14)细胞,三轮试验共获得抗性克隆30个,人14号染色体有效转移率为1×10-6.实验结果表明,人14号染色体完整的自A9细胞转移至DT40细胞,获得的DT40(#14)细胞可用于制备含人抗体基因的人类人工染色体,用于人抗体基因的表达研究.     

一种适用于生产转染色体动物的微细胞制备方法

微细胞介导的染色体转移技术(MMCT)是一项将外源染色体转入哺乳动物细胞的技术,具有广阔的应用前景.与体细胞核移植技术结合,MMCT可用于生产具有重要医学药用价值和优良农业生产性状的转染色体动物.制备高质量的微细胞是关系MMCT技术成功的关键步骤之一.通过荧光染色和吉姆萨染色分析,结果表明,A9(neo12)细胞经0.2mg/L秋水仙素酰胺处理48h后,89%的细胞产生微核化,每个细胞平均形成10个微核.微核化的细胞在含有20mg/L细胞松弛B的Percoll密度梯度介质中,经39000g高速离心后,包含微细胞、完整细胞、细胞核和细胞碎片的混合液,依次通过8μm和5μm孔径的滤膜过滤后可获得纯化的微细胞溶液.通过光学显微镜和吉姆萨染色观察,可见微细胞为一群直径约为3～5μm的类细胞核的球形物质.微细胞PCR技术首次用于检测微细胞溶液的质量,检测结果显示,所制备的溶液中均匀分布着带有目的染色体的微细胞,适用于进一步作转染色体动物实验.     

An aptasensor for ochratoxin A based on grafting of polyethylene glycol on a boron-doped diamond microcell

A novel strategy for the fabrication of an electrochemical label-free aptasensor for small-size molecules is proposed and demonstrated as an aptasensor for ochratoxin A (OTA). A long spacer chain of polyethylene glycol (PEG) was immobilized on a boron-doped diamond (BDD) microcell via electrochemical oxidation of its terminal amino groups. The amino-aptamer was then covalently linked to the carboxyl end of the immobilized PEG as a two-piece macromolecule, autoassembled at the BDD surface, forming a dense layer. Due to a change in conformation of the aptamer on the target analyte binding, a decrease of the electron transfer rate of the redox [Fe(CN)₆]^4–/3– probe was observed. To quantify the amount of OTA, the decrease of the square wave voltammetry (SWV) peak maximum of this probe was monitored. The plot of the peak maximum against the logarithm of OTA concentration was linear along the range from 0.01 to 13.2 ng/L, with a detection limit of 0.01 ng/L. This concept was validated on spiked real samples of rice.     

Multiple strategies for gene transfer, expression, knockdown, and chromatin influence in mammalian cell lines and transgenic animals

Manipulation of the eukaryotic genome has contributed to the progress in our knowledge of multicellular organisms but has also ameliorated our experimental strategies. Biological questions can now be addressed with more efficiency and reproducibility. There are new and varied strategies for gene transfer and sequence manipulation with improved methodologies that facilitate the acquisition of results. Cellular systems and transgenic animals have demonstrated their invaluable benefits. In this review, I present an overview of the methods of gene transfer with particular attention to cultured cell lines and large-scale sequence vectors, like artificial chromosomes, with the possibility of their manipulation based on homologous recombination strategies. Alternative strategies of gene transfer, including retroviral vectors, are also described and the applications of such methods are discussed. Finally, several comments are made about the influence of chromatin structure on gene expression. Recent experimental data have shown that for convenient stable transgene expression, the influence of chromatin structure should be seriously taken into account. Novel chromatin regulatory and structural elements are proposed as an alternative for proper and sustained gene expression. These chromatin elements are facing a new era in transgenesis and we are probably beginning a new generation of gene and cancer therapy vectors.     

微细胞介导的染色体转移技术及其应用

微细胞介导的染色体转移技术（MMCT）是一项利用微细胞将外源染色体转入受体细胞的技术。该技术是在细胞融合的基础之上发展起来的,是细胞融合技术的进一步细化,在当代生物的若干领域里得到了广泛的应用。～些肿瘤抑制基因、端粒酶抑制基因、诱导衰老基因以及DNA修复基因都是通过MMCT技术取得细胞内识别和定位,由此促进了针对这些基因的功能研究,并为相关疾病的治疗提供了依据。同时,MMcT技术也为其他领域如表观遗传学、基因组印迹、哺乳动物人工染色体等方面的进一步研究提供了有力的手段。与体细胞核移植技术结合,MMCT还可用于建立具有重要医学药用价值和优良农业生产性状的转染色体动物,显示其具有广阔的应用前景。本文概述了MMCT技术及其在相关领域的应用与发展趋势。