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。     

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.     

产生人抗体的转染色体动物研究进展

现已证明,应用抗体治疗疾病是一种非常成功的方法。单克隆抗体的生产使免疫治疗达到一个新水平,但鼠源单抗在治疗人体疾病方面有很多问题,而人源化抗体可以解决这些问题。目前抗体人源化已由鼠嵌合抗体发展到了转基因动物表达完全人抗体阶段,而人类人工染色体(HAC)载体的发展和微细胞介导的转染色体技术使得产生携带人类免疫球蛋白基因位点的转染色体动物成为可能。通过HAC将人的免疫球蛋白基因转入后,这类转染色体动物可以产生大量人源化多克隆抗体,这对预防及治疗疾病,甚至防御生物武器都有很重要的作用。转染色体技术可以使动物携带大而复杂的人类基因或基因簇,这些转基因动物有助于研究人类基因组在体内的功能作用,并用于各种疾病研究和生产药物蛋白。     

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。     

稀土元素钬对蚕豆的细胞毒性和遗传毒性研究

运用氧化钬与稀硝酸反应制备结晶,以去离子水溶解并且稀释成梯度溶液,对蚕豆根尖染毒6 h,分别修复培养22h和24h,观察根尖变化,统计微核率、染色体畸变率及有丝分裂指数。结果表明,4mg/L（以氧化钬质量体积浓度计）以下剂量对根尖生长具有促进作用;随着浓度的递增,微核率、染色体畸变率逐步上升,有丝分裂指数逐步下降,表现出明显的剂量-效应关系,说明稀土元素钬具有一定的细胞毒性和遗传毒性。同时,不同修复组在微核率、染色体畸变率及有丝分裂指数上也存在一定差异,表现为微核率22h修复组低于24 h 修复组,而染色体畸变率和分裂指数均高于24h修复组。微核检测应在染色体畸变检测之后进行。      

Taxol诱导CHO细胞微核化的动态分析

本实验观察到10μM Taxol对CHO细胞的G_1→S→G_2→M期的进程不产生影响,但使CHO的有丝分裂中期大大延长,不能形成纺锤体,凝集的染色体散在于胞质中,不形成中期板。经Taxol处理的CHO细胞的TM要比正常CHO细胞TM长13倍,不能完成胞质分裂,形成带有3—15个微核的细胞,最小的微核只含有1个染色体的DNA含量,微核化细胞形成率可高达96%左右。微核化细胞可进入下一个细胞周期,形成更多微核的细胞。用1×10~(-4)M二硝基苯酚(DNP)不影响微核化细胞的形成,加进10微克/毫升环己亚胺后7小时使微核化细胞形成速率减慢。     

人工雌核发育草鱼染色体倍性的鉴定

运用经典的红细胞及细胞核体积大小测量方法以及流式细胞仪,检测了人工诱导雌核发育草鱼染色体倍性与DNA含量.雌核发育草鱼红细胞体积为(333.5±41.94)μm3,细胞核体积为(20.7±2.378)μm3;与所测普通草鱼红细胞体积(343.8±50.1)μm3,细胞核体积(21.2±1.98)μm3,没有显著差异.雌核发育草鱼DNA含量(2C)平均为2.23pg,普通草鱼DNA含量(2C)2.20pg,两者无显著差异.研究结果表明,人工雌核发育草鱼与普通草鱼具有相同的染色体倍性.     

激光微束在遗传操作中的应用

激光微束是80年代后期发展起来的一种很有前途的遗传操作技术。该技术利用激光方向性好、光色单一和亮度高等独一无二的特点,把激光束通过光学系统引入显微镜并聚焦成很小的光点(直径小于1μm)。这种直径很小但功能密度很高的激光束照射细胞后,在细胞膜的表面引起可修复性的微损伤,从而改变细胞膜的通透性,因此激光微束可用于诱导基因转移、染色体切割、细胞核打孔以及细胞融合等与基因导入有关的遗传操作。激光微束技术使得对细胞或细胞器进行精细的细胞或亚细胞水平的显微外科术成为可能。     

中华鲟染色体组型的研究

材料和方法 试验鱼于1983年冬天和1984年春天取自湖北省武汉市市场(长江水系),二雌一雄,体长为2.0—3.2米,体重为99—220公斤。取肾脏组织在无菌的条件下用PBS溶液进行多次洗涤后,用剪刀剪成小块,再用0.02％胰蛋白酶消化15分钟,离心分离肾细胞,进行短期培养,用0.075M氯化钾溶液低渗处理,气干法制片,吉姆萨染色。在油镜下观察、计数、确定染色体数目。然后选择分散好、形态清晰的中期分裂相做显微镜照相,将标准的中期分裂相剪下,按同源染色体配对、测量,并用统计学方法计算其臂比和相对长度。根据Levan等(1964)提出的按着丝点的位置进行染色体的命名和分类。     

紫万年青的核型分析

本文采用直接敲片法制作紫万年青植物染色体标本,通过体细胞染色体计数确定其染色体数目为12条,核型分析表明紫万年青植物染色体总长度为57.35μm,全组染色体平均长度9.56μm,其核型公式为K(2n)=12=3m+3Sm.同时通过观察、测量发现其染色体绝对长度变异范围为11.36～7.72μm,其相对长度组成为2n=12=6M2+6M1;最长染色体与最短染色体之比为1.47:1,臂比的变异范围为1.01～2.56,臂大于2的染色体占全组染色体的33.33%,属于"2A"类型.另一方面,间期附加核型特征确定紫万年青属于复杂染色中心型.本研究将对紫万年青植物的起源、系统演化及品种改良等提供必要的细胞遗传学依据.     

The transfer of human artificial chromosomes via cryopreserved microcells

Microcell-mediated chromosome transfer (MMCT) technology enables a single and intact mammalian chromosome or megabase-sized chromosome fragments to be transferred from donor to recipient cells. The conventional MMCT method is performed immediately after the purification of microcells. The timing of the isolation of microcells and the preparation of recipient cells is very important. Thus, ready-made microcells can improve and simplify the process of MMCT. Here, we established a cryopreservation method to store microcells at −80 °C, and compared these cells with conventionally- (immediately-) prepared cells with respect to the efficiency of MMCT and the stability of a human artificial chromosome (HAC) transferred to human HT1080 cells. The HAC transfer in microcell hybrids was confirmed by FISH analysis. There was no significant difference between the two methods regarding chromosome transfer efficiency and the retention rate of HAC. Thus, cryopreservation of ready-to-use microcells provides an improved and simplified protocol for MMCT.     

A method to generate microcells from human lymphoblasts for use in microcell mediated chromosome transfer

Summary A method is described to generate microcells from human lymphobalsts for use in microcell-mediated chromosome transfer (MMCT). Micronuclei were induced in cells from a human lymphoblastic cell line by prolonged colcemid treatment, and were separated from these lymphoblasts by: (a) attaching the cells to Concanvalin A coated plastic slides designed for enucleation, and (b) centrifuging the slides in medium containing cytochalasin B. Microcells of less than 3 μm in diameter were fused with thymidine kinase negative mouse fibroblast (LMTK⁻). HAT medium (hypoxanthine, aminopterin and thymidine) was used to select microcell hybrids expressing thymidine kinase activity. Positive clones were isolated and Q-banded for chromosome analysis. Unlike previous methods this procedure permits microcells to be easily generated from lymphoid cells. This methodology of enucleation of microcells may be extended to a variety of other donor cell types which can be micronucleated but which do not adhere tightly to enucleation slides and do not exhibit extrusion subdivision. This feature makes our methodology particularly useful for constructing a library of hybrid clones containing one or a few human chromosomes.     

Isolation of a new cDNA clone encoding an Rh polypeptide associated with the Rh blood group system

We searched for a human chromosome that would restore the cholesterol metabolism in 3T3 cell lines (SPM-3T3) derived from homozygous sphingomyelinosis mice (spm/spm). Mouse A9 cells containing a single copy of pSV2neo-tagged chromosomes 9, 11, or 18 derived from normal human fibroblasts served as donor cells for transfer of human chromosomes. Purified A9 microcells were fused with SPM-3T3 cells, and the microcell hybrids were selected in medium containing G418 antibiotics. The microcell hybrids that contained human chromosomes 9, 11, or 18 in a majority of cells were examined. The accumulation of intracellular cholesterol in the microcell hybrids containing a chromosome 18 decreased markedly, whereas in the microcell hybrids containing either chromosomes 9 or 11 it was similar to that in SPM3T3 cells. The SPM-3T3 cells with an intact chromosome 18 were further passaged and subcloned. Clones which again accumulated intracellular cholesterol had concurrently lost the introduced chromosome 18. The abnormal accumulation was associated with a decrement in the esterification of exogenous cholesterol. These findings suggest that the gene responsible for the abnormal cholesterol metabolism in the spm/spm mice can be restored by a hu man chromosome 18. The gene was tentatively mapped on 18pter18p11.3 or 18q21.3qter that was lost during subcloning, thereby resulting in reaccumulation of the intracellular cholesterol.     

Introduction of new genetic markers on human chromosomes

The purpose of this study was to use DNA transfection and microcell chromosome transfer techniques to engineer a human chromosome containing multiple biochemical markers for which selectable growth conditions exist. The starting chromosome was a t(X;3)(3pter----3p12::Xq26----Xpter) chromosome from a reciprocal translocation in the normal human fibroblast cell line GM0439. This chromosome was transferred to a HPRT (hypoxanthine phosphoribosyltransferase)-deficient mouse A9 cell line by microcell fusion and selected under growth conditions (HAT medium) for the HPRT gene on the human t(X;3) chromosome. A resultant HAT-resistant cell line (A9(GM0439)-1) contained a single human t(X;3) chromosome. In order to introduce a second selectable genetic marker to the t(X;3) chromosome, A9(GM0439)-1 cells were transfected with pcDneo plasmid DNA. Colonies resistant to both G418 and HAT medium (G418r/HATr) were selected. To obtain A9 cells that contained a t(X;3) chromosome with an integrated neo gene, the microcell transfer step was repeated and doubly resistant cells were selected. G418r/HATr colonies arose at a frequently of 0.09 to 0.23 x 10(-6) per recipient cell. Of seven primary microcell hybrid clones, four yielded G418r/HATr clones at a detectable frequency (0.09 to 3.4 x 10(-6)) after a second round of microcell transfer. Doubly resistant cells were not observed after microcell chromosome transfers from three clones, presumably because the markers were on different chromosomes. The secondary G418r/HATr microcell hybrids contained at least one copy of the human t(X;3) chromosome and in situ hybridization with one of these clones confirmed the presence of a neo-tagged t(X;3) human chromosome. These results demonstrate that microcell chromosome transfer can be used to select chromosomes containing multiple markers.     

Exploitation of the interaction of measles virus fusogenic envelope proteins with the surface receptor CD46 on human cells for microcell-mediated chromosome transfer

Background  

Microcell-mediated chromosome transfer (MMCT) is a technique by which a chromosome(s) is moved from donor to recipient cells by microcell fusion. Polyethylene glycol (PEG) has conventionally been used as a fusogen, and has been very successful in various genetic studies. However, PEG is not applicable for all types of recipient cells, because of its cell type-dependent toxicity. The cytotoxicity of PEG limits the yield of microcell hybrids to low level (10^-6 to 10^-5 per recipient cells). To harness the full potential of MMCT, a less toxic and more efficient fusion protocol that can be easily manipulated needs to be developed.     

Microcell-mediated chromosome transfer (MMCT): small cells with huge potential

Microcell-mediated chromosome transfer (MMCT) is a technique that has been in use since the 1970s for the fusion of microcells, containing single or a small number of chromosomes, with whole cells, and the subsequent selection of the hybrids. MMCT can be carried out with somatic cells, embryonic carcinoma (EC) or embryonic stem (ES) cell recipients, to study in vitro or in vivo effects of the transferred genetic material. These effects may be unpredictable–do the transferred genes function normally while in the regulatory milieu of the host cell? Will epigenetic effects become apparent, and how will these alter gene expression? What happens to the host cell phenotype? Here, we present a review of MMCT in which we argue that, although this is an old technique, its adaptability and efficiency make it an excellent method for the dissection of gene function and dysfunction in a very wide range of current systems.     

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.     

Construction of microcell hybrid clones containing specific mouse chromosomes: application to autosomes 8 and 17.

Fibroblast cultures prepared from mice homozygous for a Robertsonian translocation (centric fusion) between autosomes 8 and 17 [Rb(8.17)] were used as donors in microcell-mediated chromosome transfer experiments. By using hamster recipient cells deficient in adenine phosphoribosyltransferase (APRT-) and selecting for expression of murine APRT (a chromosome 8 marker), microcell hybrids were isolated which retained only the mouse Rb(8.17) translocation in addition to the hamster chromosome complement. The translocation was stable in cells maintained under APRT+ selective pressure, and mouse marker traits encoded by genes on both chromosomes 8 and 17 segregated concordantly. A second family of hybrid clones was constructed by fusing microcells derived from wild-type mouse fibroblasts with APRT- hamster cells. Four of six clones analyzed retained only mouse chromosome 8. These studies demonstrated that microcell hybrids containing specific Robertsonian translocations as the only donor-derived genetic material can be obtained. Furthermore, a number of Robertsonian translocations between chromosomes which carry selectable markers (chromosomes 3, 8, and 11) and other autosomes have been described. By using fibroblast cultures prepared from mice containing these translocations as donors in microcell fusions, 18 of the 20 mouse chromosomes could be selectively fixed in different hybrid clones. Thus, a collection of 20 hybrid clones, each containing a single, specific mouse chromosome, can be constructed by using the strategy described in this report. The potential utility of such a monochromosomal hybrid panel is discussed.