微囊介导的染色体转移技术及其应用

微囊介导的染色体转移技术是从细胞融合技术演变而来的,它是把供体细胞用秋水仙胺和细胞松弛素B处理,制备成含有一条或几条染色体的微囊,再与受体细胞融合的技术.     

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.     

用微细胞技术转移鱼类遗传因子初报

自Ege等(1974)4的工作开始,在70年代已逐步建立起一种通过微细胞把一个细胞的染色体转移到另一个细胞中,并作为在杂种细胞里起作用的遗传因素,永远地保留下去的技术。       

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.     

由质粒介导的浑球红假单胞菌的染色体基因转移

通过接合转移,质粒pR751：：813、pACYCl84：：Tn5和RP4：：Mu从大肠杆菌引入浑球红假单胞菌。以质粒pR751：：813和RPI：：TnS01为介导的染色体转移,构威了一条染色体基因连锁图,并确定了包括半胱氨酸和胞嘧啶在内的六个遗传标记的相对位置。决定卡那霉素抗性的转座子Tn5以每供体}．3×10一’频率从大肠杆菌转入浑球红假单胞菌。约1．5％的“m‘分离物是营养缺陷型。形成的半胱氨酸变种是稳定的,回复突变频率约为4×10-10。转座子插入诱变产生的变种可用于遗传分析研究。     

鱼类微细胞和小分离细胞制备技术的研究

本文以3种鱼类组织细胞系为材料,对鱼类微细胞和小分离细胞制备技术及其形成机制进行了研究。在微细胞制备中,观察了细胞的微核化过程,微核是由间期状态的细胞核不规则分裂而成。光镜和扫描电镜观察表明,在小分离细胞聚集体形成过程中,细胞核的变化与微细胞的微核化过程相一致,但其细胞质的分裂机制不同,当细胞质进行异常分裂的同时,细胞的微核也随同细胞质不规则分裂而被分配至各个小分离细胞内。     

蚕豆花粉母细胞染色体畸变同染色质胞间转移的关系

采用压碎、石蜡切片及植物染色体分带技术观察蚕豆花粉母细胞减数分裂过程发现:1.蚕豆花粉母细胞减数分裂的凝线期也出现染色质胞间转移现象;2.在8.08%的花粉母细胞内,染色体偏离正常的数目(n=6);3.染色体结构也有显著改变,其中包括染色体断裂、桥和长度的增减;4.出现双核和无核的花粉母细胞。大量证据表明,这些畸变现象同凝线期的染色质胞间转移有关。     

电穿透技术在细胞融合和基因转移中的应用

按下电钮,能使一对电极间的细胞融合,还能使基因导入细胞,这是八十年代生物技术中引人注目的新成就之一.电子技术以其精确和简便之特长,又一次推动了生命科学的技术进步.几年前,西德的Zimmermann和Neumann先后报道了分别称作细胞电融合     

百合花粉母细胞间细胞融合期间腺苷三磷酸酶活性的细胞化学定位及其与染色质胞间转移的关系

用标准的磷酸铅沉淀的细胞化学方法,对百合花粉母细胞间染色质穿壁运动期间及其前后三个时期中的腺苷三磷酸酶(ATP 酶)活性进行了超微结构的定位。结果表明:(1)在穿壁前,ATP 酶活性主要定位于质膜、胞间连丝及细胞间隙;在内质网、高尔基体、质体和某些局部的基质(groundplasm)中,也表现有 ATP 酶活性反应的产物;但在染色质和核仁中,一般都没有这种反应。(2)在穿壁时,染色质从一个细胞穿壁转移到另一个相邻细胞,同时看到染色质和核仁内出现密集的 ATP 酶活性反应产物;在内质网和高尔基体的腔内以及质体的片层上也产生明显的 ATP 酶活性反应;而在质膜、胞间连丝及细胞间隙内 ATP 酶活性明显降低,甚至看不到明显的活性反应。(3)在穿壁后,质膜及细胞间隙中又产生明显的 ATP 酶活性反应产物,但核内染色质上的 ATP 酶活性则显著降低,而核仁内则仍有较高的活性。同前二个时期一样,内质网、高尔基体和质体上的 ATP 酶仍表现明显的活性反应。最后讨论了三个不同发育时期 ATP 酶活性及其分布部位的改变与染色质胞间转移的关系。     

Generation of a novel isogenic trisomy panel in human embryonic stem cells via microcell-mediated chromosome transfer

Aneuploidy is the gain or loss of a chromosome. Down syndrome or trisomy (Ts) 21 is the most frequent live-born aneuploidy syndrome in humans and extensively studied using model mice. However, there is no available model mouse for other congenital Ts syndromes, possibly because of the lethality of Ts in vivo, resulting in the lack of studies to identify the responsible gene(s) for aneuploid syndromes. Although induced pluripotent stem cells derived from patients are useful to analyse aneuploidy syndromes, there are concerns about differences in the genetic background for comparative studies and clonal variations. Therefore, a model cell line panel with the same genetic background has been strongly desired for sophisticated comparative analyses. In this study, we established isogenic human embryonic stem (hES) cells of Ts8, Ts13, and Ts18 in addition to previously established Ts21 by transferring each single chromosome into parental hES cells via microcell-mediated chromosome transfer. Genes on each trisomic chromosome were globally overexpressed in each established cell line, and all Ts cell lines differentiated into all three embryonic germ layers. This cell line panel is expected to be a useful resource to elucidate molecular and epigenetic mechanisms of genetic imbalance and determine how aneuploidy is involved in various abnormal phenotypes including tumourigenesis and impaired neurogenesis.     

The manipulation of chromosomes by mankind: the uses of microcell-mediated chromosome transfer

Microcell-mediated chromosome transfer (MMCT) was a technique originally developed in the 1970s to transfer exogenous chromosome material into host cells. Although, the methodology has not changed considerably since this time it is being used to great success in progressing several different fields in modern day biology. MMCT is being employed by groups all over the world to hunt for tumour suppressor genes associated with specific cancers, DNA repair genes, senescence-inducing genes and telomerase suppression genes. Some of these genomic discoveries are being investigated as potential treatments for cancer. Other fields have taken advantage of MMCT, and these include assessing genomic stability, genomic imprinting, chromatin modification and structure and spatial genome organisation. MMCT has also been a very useful method in construction and manipulation of artificial chromosomes for potential gene therapies. Indeed, MMCT is used to transfer mainly fragmented mini-chromosome between cell types and into embryonic stem cells for the construction of transgenic animals. This review briefly discusses these various uses and some of the consequences and advancements made by different fields utilising MMCT technology. Review related to the 15th International Chromosome Conference (ICC XV), held in September 2004, Brunel University, London, UK     

Determination of the chromosomal site for the human radiosensitive ataxia telangiectasia gene by chromosome transfer

The chromosomal localization of the gene which complements radiation hypersensitivity of AT cells was studied by microcell-mediated chromosome transfer. A 6-thioguanine-resistant derivative of an immortalized AT cell line, AT2KYSVTG, was used as a recipient for microcell-mediated chromosome transfer from 4 strains of mouse A9 cells, 3 of which carried a human X/11 recombinant chromosome containing various regions of chromosome 11, while the other carried an intact X chromosome. HAT-resistant microcell hybrids were isolated and examined for their radiosensitivity and chromosome constitution. The microcell hybrid clones obtained from the transfer of an intact X chromosome or an X/11 chromosome bearing the pter → q13 region of chromosome 11 did not show a difference in radiosensitivity from parental AT cells, while those obtained from the transfer of X/11 chromosomes bearing either the p11 → qter or the pter → q23 region of chromosome 11 exhibited a marked radioresistance which was comparable to normal human fibroblasts. A HAT-resistant but radiosensitive variant was further obtained from the microcell fusion with an A9 cell strain carrying an X/11 chromosome bearing the 11p11 → qter region, in which a deletion at the 11q23 region was found. The results indicate that the gene which complements a radiosensitive phenotype of AT is located at the q23 region of chromosome 11.     

Understanding the X chromosome inactivation cycle in mice: A comprehensive view provided by nuclear transfer

During mouse development, imprinted X chromosome inactivation (XCI) is observed in preimplantation embryos and is inherited to the placental lineage, whereas random XCI is initiated in the embryonic proper. Xist RNA, which triggers XCI, is expressed ectopically in cloned embryos produced by somatic cell nuclear transfer (SCNT). To understand these mechanisms, we undertook a large-scale nuclear transfer study using different donor cells throughout the life cycle. The Xist expression patterns in the reconstructed embryos suggested that the nature of imprinted XCI is the maternal Xist-repressing imprint established at the last stage of oogenesis. Contrary to the prevailing model, this maternal imprint is erased in both the embryonic and extraembryonic lineages. The lack of the Xist-repressing imprint in the postimplantation somatic cells clearly explains how the SCNT embryos undergo ectopic Xist expression. Our data provide a comprehensive view of the XCI cycle in mice, which is essential information for future investigations of XCI mechanisms.     

Plant artificial chromosome technology and its potential application in genetic engineering

Genetic engineering with just a few genes has changed agriculture in the last 20 years. The most frequently used transgenes are the herbicide resistance genes for efficient weed control and the Bt toxin genes for insect resistance. The adoption of the first‐generation genetically engineered crops has been very successful in improving farming practices, reducing the application of pesticides that are harmful to both human health and the environment, and producing more profit for farmers. However, there is more potential for genetic engineering to be realized by technical advances. The recent development of plant artificial chromosome technology provides a super vector platform, which allows the management of a large number of genes for the next generation of genetic engineering. With the development of other tools such as gene assembly, genome editing, gene targeting and chromosome delivery systems, it should become possible to engineer crops with multiple genes to produce more agricultural products with less input of natural resources to meet future demands.     

The microcell-mediated transfer of human chromosome 8 restores the deficient N-acetylytransferase activity in skin fibroblasts of Mucopolysaccharidosis type IIIC patients

The candidate gene for Mucopolysaccharidosis (MPS) type IIIC has been localized to the pericentric region of the chromosome 8 by the linkage disequilibrium analysis. To validate the localization of the gene, we rescued the deficient acetyl-coenzyme A: alpha-glucosaminide-N-acetylytransferase activity in the cultured cells of MPS IIIC patients by functional complementation via microcell-mediated chromosome transfer. The introduction of the target human monochromosome completely restored the activity confirming functional localization of the candidate gene on human chromosome 8.     

Cytogenetics and the major histocompatibility complex in a proliferating microcell-mediated hybrid clone

Summary By way of a microcell fusion, three chromosomes from a B82HTQ2 (TK^–) cell were introduced into a PG19 (HGPRT^–) cell. Analysis of this hybrid clone showed that the transferred chromosomes restored a positive HGPRT status but failed to produce heterozygosity for the major histocompatibility complex (H-2). The three chromosomes also proved stable in both long term culture in vitro and tumor testing in mice. It is suggested that the method could prove useful in correcting genetic defects or in introducing new genetic characteristics without the introduction of the genes coding for major histocompatibility antigens. The surface structure of the microcells was studied by scanning electron microscope. The optimum for induction of the microcells from B82HTQ2 cells and its' purification were reported here. Frequency of the sister chromatid exchange (SCE) of the hybrid cells and their sensitivity to mitomycin C (MMC) were also examined.