CAJ | 학술논문

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0.2 mg/L秋水仙素酰胺处理48 h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20 mg/L细胞松弛B的Percoll密度梯度介质中,经39 000g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8 μm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.
미세포개도적염색체전이기술(MMCT)시일항장외원염색체전입포유동물세포적기술,구유엄활적응용전경.여체세포핵이식기술결합,MMCT가용우생산구유중요의학약용개치화우량농업생산성상적전염색체동물.제비고질량적미세포시관계MMCT기술성공적관건보취지일.통과형광염색화길모살염색분석,결과표명,A9(neo12)세포경0.2 mg/L추수선소선알처리48 h후,89%적세포산생미핵화,매개세포평균형성10개미핵.미핵화적세포재함유20 mg/L세포송이B적Percoll밀도제도개질중,경39 000g고속리심후,포함미세포、완정세포、세포핵화세포쇄편적혼합액,의차통과8 μm화5μm공경적려막과려후가획득순화적미세포용액.통과광학현미경화길모살염색관찰,가견미세포위일군직경약위3～5μm적류세포핵적구형물질.미세포PCR기술수차용우검측미세포용액적질량,검측결과현시,소제비적용액중균균분포착대유목적염색체적미세포,괄용우진일보작전염색체동물실험.
Microcell mediated chromosome transfer (MMCT) is a challenging technique for introducing exogenous chromosomes into interested mammalian cells. Combined with the somatic cell nuclear transfer technique, MMCT has been employed for producing transchromosomic animals of medical and agricultural value. Producing high quality of microcells is a key step in the success of MMCT. Eamamined by fluorescin staining and Giemsa staining, 0.2 mg/L colcemid was considered suitable for inducing high percentage of micronuclei in A9 (neo12) cells, without causing death of a mass of cells. Microcells were produced by centrifugation of micronucleated A9 (neo12) cells in Percoll density gradient containing 20 mg/L Cytochalasin B at 39 000 g. The resulting mixture of microcells, whole cells, karyoplasts and cytoplast fragments was filtered through 8 μm and 5 μm size membrane pores sequentially to obtain pure preparation of microcells. Microcells were then characterized by Giemsa staining and microcell PCR was first applied for examination of the quality of microcell preparation. The result showed that microcells containing our interest chromosomes-human chromosome 12 were equally distributed in the preparation, the preparation was suitable for use in generation of transchromosomic animals.