deJong G, Telenius A H, Telenius H, Perez C F, Drayer J I, Hadlaczky G
Chromos Molecular Systems, Inc., Vancouver, British Columbia, Canada.
Cytometry. 1999 Feb 1;35(2):129-33.
A pilot production facility has been established to isolate mammillian artificial chromosomes at high purity by using flow cytometric techniques. Dicentric chromosomes have been generated by the targeted amplification of pericentric heterochromatic and centromeric DNA by activating the "megareplicator." Breakage of these dicentric chromosomes generates satellite DNA-based artificial chromosomes (SATAC) from 60 to 400 megabases.
For large-scale production, we have developed cell lines capable of carrying one or two SATACs. A SATAC, because of a high adenine-thymine (AT) composition, is easily identified and sorted by using chromomycin A3 and Hoechst 33258 stains and a dual laser high-speed flow cytometer. A prototype SATAC (60 megabases) has been characterized. The prototype SATAC has been isolated from an original rodent/human hybrid cell line and transferred by using modified microcell fusion into a CHO production cell line.
Metaphase chromosomes from this production cell line were isolated in a modified polyamine buffer, stained, and sorted by using a modified sheath buffer that maintains condensed chromosomes. SATACs are routinely sorted at rates greater than 1 million per hour. Sorted SATACs have been transferred to a variety of cells by using microcell fusion technology and were found to be functional.
By developing new SATAC containing cell lines with fewer numbers of chromosomes in conjunction with operating a high speed flow sorter we have effectively generated an efficient production facility geared purely for the isolation of SATACs.
已建立一个中试生产设施，通过流式细胞术技术高纯度分离哺乳动物人工染色体。通过激活“超级复制子”对着丝粒周围异染色质和着丝粒DNA进行靶向扩增，已产生双着丝粒染色体。这些双着丝粒染色体的断裂产生了大小从60到400兆碱基的基于卫星DNA的人工染色体（SATAC）。
为了大规模生产，我们开发了能够携带一个或两个SATAC的细胞系。由于SATAC具有高腺嘌呤-胸腺嘧啶（AT）组成，使用放线菌素A3和Hoechst 33258染色以及双激光高速流式细胞仪很容易识别和分选。已对一个原型SATAC（60兆碱基）进行了表征。该原型SATAC已从原始啮齿动物/人类杂交细胞系中分离出来，并通过改良的微细胞融合转移到CHO生产细胞系中。
从该生产细胞系中分离出中期染色体，在改良的多胺缓冲液中染色，并使用能维持染色体浓缩的改良鞘液进行分选。SATAC通常以每小时超过100万个的速度进行分选。分选后的SATAC已通过微细胞融合技术转移到多种细胞中，并发现其具有功能。
通过开发含染色体数量较少的新型SATAC细胞系并结合操作高速流式分选仪，我们有效地建立了一个专门用于分离SATAC的高效生产设施。
