Lucas J J, Kates J R
Cell. 1976 Mar;7(3):397-405. doi: 10.1016/0092-8674(76)90169-0.
Using the mouse L-cell line as a model system, a generalized approach is presented for nuclear transplantation in cultured cells resulting in the construction of cytoplasmic-nuclear hybrid cells. Techniques were developed for the preparation of cytoplast and karyoplasts having minimum contamination by parent whole cells. Sendai virusmediated fusion was performed in a manner which maximized the formation of the desired fusion products-cells having one cell equivalent of cytoplasm from one parent and a nucleus from a second parent. The viability of the fusion products was established by examination of photographic records of the developing cultures. Using these techniques, we found that nuclei could be introduced routinely into 10-30% of a cytoplast culture. From determinations of the increase in cell number with time, it was estimated that at least 30% of the reconstructed cells were capable of division. The approach was next applied to the formation of hybrid cells from L-cell cytoplasts and A9 cell karyoplasts. The A9 cell line is an azaguanine-resistant derivative of L cells. Thus any whole cells remaining in the culture of fused cells were readily eliminated by treatment with the purine analogue. The culture of remaining cytoplasmic-nuclear hybrid cells grew to confluence in the presence of azaguanine. The applicability of the approach to the construction of hybrid cells using parent lines from different organisms is briefly discussed.
以小鼠L细胞系作为模型系统，本文提出了一种在培养细胞中进行核移植的通用方法，用于构建细胞质-细胞核杂种细胞。开发了一些技术，用于制备受亲本全细胞污染最小的胞质体和核体。仙台病毒介导的融合以一种方式进行，这种方式能使所需融合产物——具有来自一个亲本的一个细胞当量细胞质和来自第二个亲本的一个细胞核的细胞——的形成最大化。通过检查发育中的培养物的照片记录来确定融合产物的活力。使用这些技术，我们发现细胞核可以常规地导入到10%-30%的胞质体培养物中。通过测定细胞数量随时间的增加，估计至少30%的重建细胞能够分裂。接下来，该方法被应用于由L细胞胞质体和A9细胞核体形成杂种细胞。A9细胞系是L细胞的抗氮杂鸟嘌呤衍生物。因此，通过用嘌呤类似物处理，可以很容易地消除融合细胞培养物中残留的任何全细胞。残留的细胞质-细胞核杂种细胞在氮杂鸟嘌呤存在的情况下生长至汇合。简要讨论了该方法在使用来自不同生物体的亲本系构建杂种细胞方面的适用性。
