Robl J M, Prather R, Barnes F, Eyestone W, Northey D, Gilligan B, First N L
J Anim Sci. 1987 Feb;64(2):642-7. doi: 10.2527/jas1987.642642x.
This study was conducted to develop a method for transplanting nuclei in bovine embryos and to test the development of several stages of donor nuclei transplanted to enucleated pronuclear recipient embryos. Pronuclear embryos were centrifuged to reveal nuclei. Nuclei were removed without penetrating the plasma membrane as membrane-bound karyoplasts, and were inserted into enucleated zygotes by electrically induced cell fusion. The highest rate of fusion (79%) occurred in Zimmerman Cell Fusion medium at 100 V for 20 to 40 microseconds with the fusion membranes oriented parallel to the electrodes. The effect of nuclear transplantation on development was tested in pronuclear embryos in which nuclei were removed and reinserted and the embryos were then transferred to sheep oviducts for 5 d. Of the intact nuclear transplant embryos recovered, 5/29 (17%) developed to morulae or blastocysts compared with 11/30 (37%) of the non-manipulated embryos. Two nuclear transplant embryos were transferred to a recipient cow, and both developed to normal offspring. When nuclei from two-, four-, or eight-cell embryos were transplanted to pronuclear recipient embryos, no development was observed.
本研究旨在开发一种牛胚胎细胞核移植方法，并测试移植到去核原核受体胚胎中的供体细胞核在几个发育阶段的发育情况。对原核胚胎进行离心以显示细胞核。将细胞核作为膜结合核质体在不穿透质膜的情况下取出，并通过电诱导细胞融合将其插入去核合子中。在齐默尔曼细胞融合培养基中，融合膜与电极平行，在100 V下处理20至40微秒时，融合率最高（79%）。在去除细胞核并重新插入细胞核的原核胚胎中测试了核移植对发育的影响，然后将胚胎转移到绵羊输卵管中5天。回收的完整核移植胚胎中，5/29（17%）发育到桑葚胚或囊胚，而非操作胚胎为11/30（37%）。将两个核移植胚胎移植到一头受体母牛体内，二者均发育为正常后代。当将二细胞、四细胞或八细胞胚胎的细胞核移植到原核受体胚胎中时，未观察到发育情况。
