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通过微细胞融合构建含有一条用新霉素抗性基因标记的单个人类染色体的小鼠A9克隆。

Construction of mouse A9 clones containing a single human chromosome tagged with neomycin-resistance gene via microcell fusion.

作者信息

Koi M, Shimizu M, Morita H, Yamada H, Oshimura M

机构信息

Laboratory of Cytogenetics, Kanagawa Cancer Center Research Institute, Yokohama.

出版信息

Jpn J Cancer Res. 1989 May;80(5):413-8. doi: 10.1111/j.1349-7006.1989.tb02329.x.

DOI:10.1111/j.1349-7006.1989.tb02329.x
PMID:2502516
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC5917765/
Abstract

Normal human fibroblasts (MRC-5 or NTI-4) were transfected with pSV2-neo plasmid DNA. Fifty G418-resistant fibroblast clones were isolated and independently fused to mouse A9 cells. The cell hybrids were selected and isolated in the medium containing G418 plus ouabain. Since micronuclei were more efficiently induced in these hybrids compared to parental human fibroblasts by colcemid treatment, the transfer of neo-tagged human chromosomes in the hybrids to mouse A9 cells was performed via microcell fusion. Two hundred A9 microcell hybrids were isolated and karyotyped. Among them, thirteen microcell clones, each containing a single human chromosome 1, 2, 5, 6, 7, 8, 10, 11, 12, 15, 18, 19 or 20 were established. Isozyme analyses conformed the presence of each human chromosome in these A9 microcell clones. The results of Southern blot and chromosomal in situ hybridization analyses indicate that the human chromosomes in these clones were tagged with pSV2-neo plasmid DNA.

摘要

用pSV2-neo质粒DNA转染正常人成纤维细胞（MRC-5或NTI-4）。分离出50个对G418有抗性的成纤维细胞克隆，并将其分别与小鼠A9细胞融合。在含有G418加哇巴因的培养基中选择并分离细胞杂种。由于与亲代人成纤维细胞相比，秋水仙酰胺处理在这些杂种中更有效地诱导了微核，因此通过微细胞融合将杂种中带有neo标记的人染色体转移到小鼠A9细胞中。分离出200个A9微细胞杂种并进行核型分析。其中，建立了13个微细胞克隆，每个克隆含有一条单独的人染色体1、2、5、6、7、8、10、11、12、15、18、19或20。同工酶分析证实了这些A9微细胞克隆中每条人染色体的存在。Southern印迹和染色体原位杂交分析结果表明，这些克隆中的人染色体用pSV2-neo质粒DNA进行了标记。

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