Illmensee K, Hoppe P C, Croce C M
Proc Natl Acad Sci U S A. 1978 Apr;75(4):1914-8. doi: 10.1073/pnas.75.4.1914.
Mouse teratocarcinoma cells from the OTT6050 ascites tumor were established in tissue culture and selected for 5-bromodeoxyuridine (BrdUrd) resistance. The embryonal carcinoma cells grew without a feeder layer, remained deficient for thymidine kinase (EC 2.7.1.75), and differentiated like the original tumor into various tissues after subcutaneous injection into 129 mice. We fused the BrdUrd-resistant mouse teratocarcinoma cells with HT1080-6TG human diploid fibrosarcoma cells deficient in hypoxanthine phosphoribosyltransferase (EC 2.4.2.8) and selected for hybrid cells in hypoxanthine/aminopterin/thymidine medium. The resulting hybrid cells segregated human chromosomes quickly and retained one to three human chromosomes including chromosome 17 that carries the human genes for thymidine kinase and galactokinase (EC 2.7.1.6). Single hybrid cells from five independent clones containing human chromosome 17 were injected into mouse blastocysts bearing several genetic markers that affect the coat color phenotype and strain-specific enzyme variants in order to detect tissue differentiation derived from the injected cells. After the injection of single hybrid cells into a total of 103 experimental blastocysts that had been surgically transferred to pseudopregnant foster mothers, 49 mice were born and 2 of them clearly revealed coat mosaicism. In 2 of 17 mice thus far analyzed, the injected hybrid cells proved to be capable of participating substantially in development of seven different organs. However, human gene products have not yet been detected unequivocally in those tissues and weak human-specific galactokinase activity could be recovered only from two mosaic tissues. Our results demonstrate that, after in vitro culture and selection, at least some of the human-mouse hybrid cells still retain their in vivo potential to differentiate and become functionally integrated in the living organism. It now seems feasible to cycle mouse teratocarcinoma cells carrying human genetic material through mice via blastocyst injection to study human gene expression during differentiation.
从OTT6050腹水瘤中获取的小鼠畸胎瘤细胞在组织培养中建立，并筛选出对5-溴脱氧尿苷（BrdUrd）具有抗性的细胞。胚胎癌细胞在无饲养层的情况下生长，仍然缺乏胸苷激酶（EC 2.7.1.75），并且在皮下注射到129小鼠体内后，会像原始肿瘤一样分化为各种组织。我们将抗BrdUrd的小鼠畸胎瘤细胞与缺乏次黄嘌呤磷酸核糖基转移酶（EC 2.4.2.8）的HT1080-6TG人二倍体纤维肉瘤细胞融合，并在次黄嘌呤/氨基蝶呤/胸腺嘧啶核苷培养基中筛选杂交细胞。所得杂交细胞迅速分离出人类染色体，并保留一到三条人类染色体，包括携带人类胸苷激酶和半乳糖激酶基因（EC 2.7.1.6）的17号染色体。来自五个含有人类17号染色体的独立克隆的单个杂交细胞被注射到带有几种影响毛色表型和品系特异性酶变体的遗传标记的小鼠囊胚中，以便检测源自注射细胞的组织分化。在将单个杂交细胞注射到总共103个已通过手术移植到假孕代孕母鼠体内的实验性囊胚中后，出生了49只小鼠，其中2只明显表现出毛色嵌合体。在目前分析的17只小鼠中的2只中，注射的杂交细胞被证明能够大量参与七个不同器官的发育。然而，在这些组织中尚未明确检测到人类基因产物，并且仅从两个嵌合体组织中回收了微弱的人类特异性半乳糖激酶活性。我们的结果表明，经过体外培养和筛选后，至少一些人鼠杂交细胞仍然保留其在体内分化并在生物体中实现功能整合的潜力。现在看来，通过囊胚注射使携带人类遗传物质的小鼠畸胎瘤细胞在小鼠体内循环，以研究分化过程中的人类基因表达似乎是可行的。
