Saitoh Issei, Sato Masahiro, Iwase Yoko, Inada Emi, Nomura Toshiki, Akasaka Eri, Yamasaki Youichi, Noguchi Hirofumi
Department of Pediatric Dentistry, Kagoshima University Graduate School of Medical and Dental Sciences , Sakuragaoka, Kagoshimashi, Kagoshima , Japan.
† Section of Gene Expression Regulation, Frontier Science Research Center, Kagoshima University , Korimoto, Kagoshima, Kagoshima , Japan.
Cell Med. 2012 May 8;3(1-3):97-102. doi: 10.3727/215517912X639414. eCollection 2012 Jan.
Feeder cells are generally required for establishment and maintenance of embryonic stem (ES)/induced pluripotent stem (iPS) cells. Increased demands for generation of those cells carrying various types of vectors (i.e., KO vectors and transgenes) also require feeder cells that confer resistance to any types of preexisting selective drugs. Unfortunately, the use of the feeders that are resistant to various drugs appears to be limited to a few laboratories. Here we generated a set of gene-engineered STO feeder cells that confer resistance to several commercially available drugs. The STO cells, which have long been used as a feeder for mouse ES and embryonal carcinoma (EC) cells, were transfected with pcBIH [carrying bleomycin resistance gene () and hygromycin B phosphotransferase gene ()], pcBIP [carrying and puromycin resistance gene ()], or pcBSN [carrying and neomycin resistance gene ()]. The resulting stably transfectants (termed SHB for pcBIH, SPB for pcBIP, and SNB for pcBSN) exhibited bleomycin/hygromycin, bleomycin/puromycin, or bleomycin/neomycin, as expected. The morphology of these cells passaged over 18 generations was indistinguishable from that of parental STO cells. Of isolated clones, the SHB3, SPB3, and SNB2 clones successfully supported the growth of mouse ES cells in an undifferentiated state, when coculture was performed. PCR analysis revealed the presence of the selective markers in these clones, as expected. These SHB3, SPB3, and SNB2 cells will thus be useful for the acquisition and maintenance of genetically manipulated ES/iPS cells.
胚胎干细胞（ES）/诱导多能干细胞（iPS）的建立和维持通常需要饲养层细胞。对携带各种类型载体（即敲除载体和转基因）的这些细胞的需求增加，也需要对任何类型的现有选择性药物具有抗性的饲养层细胞。不幸的是，对各种药物具有抗性的饲养层细胞的使用似乎仅限于少数实验室。在这里，我们生成了一组基因工程化的STO饲养层细胞，它们对几种市售药物具有抗性。长期以来一直用作小鼠ES细胞和胚胎癌细胞（EC）饲养层的STO细胞，用pcBIH（携带博来霉素抗性基因（）和潮霉素B磷酸转移酶基因（））、pcBIP（携带和嘌呤霉素抗性基因（））或pcBSN（携带和新霉素抗性基因（））进行转染。所得的稳定转染子（分别称为pcBIH的SHB、pcBIP的SPB和pcBSN的SNB）如预期那样表现出对博来霉素/潮霉素、博来霉素/嘌呤霉素或博来霉素/新霉素的抗性。传代超过18代的这些细胞的形态与亲代STO细胞的形态没有区别。在分离的克隆中，当进行共培养时，SHB3、SPB3和SNB2克隆成功支持了未分化状态的小鼠ES细胞的生长。PCR分析显示这些克隆中存在选择性标记，如预期那样。因此，这些SHB3、SPB3和SNB2细胞将有助于获取和维持基因操作的ES/iPS细胞。
