Speksnijder G, Ivarie R
Department of Genetics, University of Georgia, Athens 30602-7223, USA.
Poult Sci. 2000 Oct;79(10):1430-3. doi: 10.1093/ps/79.10.1430.
Stage X chick blastoderms following oviposition were accessed via a small window in the egg. Windowing, however, substantially reduces the hatchability of eggs containing early embryos. For example, only 32 of 389 (8.2%) eggs hatched after standard windowing with or without irradiation or injection. Ex ovo culture systems can overcome this problem but are labor intensive. A modification of a standard windowing technique has yielded an average hatch rate of 32% for 892 windowed eggs independent of incubator type, gamma-irradiation, or injection of the embryo. This was a fourfold increase over a standard windowing method. Similar hatch rates were observed using fertile eggs from five chicken lines [Barred Plymouth Rock (BR), Athens-Canadian (AC), Line 0, SPAFAS, and commercial White Leghorns (WL)]. The modification involves covering the egg shell membrane with PBS after grinding away the shell and before piercing the membrane. The window is then sealed by overlaying with fresh shell membrane and cementing it in place once it has dried. The method has been used successfully for the production of somatic and germline chimeras because donor BR blastodermal cells injected into Stage X, gamma-irradiated recipient embryos from WL or AC yielded a hatch of 33.7%, of which 42.3% were feather chimeras. Two of 11 cockerels tested were germline mosaics bearing at least 1% BR sperm. The modified windowing technique may be broadly applicable in emerging technologies in avian transgenesis and development.
产卵后的X期鸡胚盘可通过在鸡蛋上开一个小窗口来获取。然而，开窗操作会大幅降低含有早期胚胎的鸡蛋的孵化率。例如，在进行有或没有辐射或注射的标准开窗操作后，389枚鸡蛋中只有32枚（8.2%）孵化成功。体外培养系统可以克服这个问题，但劳动强度大。对标准开窗技术进行改进后，892枚开窗鸡蛋的平均孵化率达到了32%，且与孵化器类型、伽马辐射或胚胎注射无关。这比标准开窗方法提高了四倍。使用来自五个鸡品系[横斑洛克鸡（BR）、雅典-加拿大鸡（AC）、0系、SPAFAS和商业白来航鸡（WL）]的受精蛋时，观察到了相似的孵化率。改进方法包括在磨去蛋壳后、刺破壳膜之前，用磷酸盐缓冲液（PBS）覆盖蛋壳膜。然后用新鲜的壳膜覆盖窗口，并在其干燥后用水泥固定到位来密封窗口。该方法已成功用于体细胞和种系嵌合体的制备，因为将供体BR胚盘细胞注射到经伽马辐射的X期WL或AC受体胚胎中，孵化率为33.7%，其中42.3%为羽毛嵌合体。在测试的11只公鸡中，有两只为种系嵌合体，携带至少1%的BR精子。改进后的开窗技术可能广泛适用于禽类转基因和发育的新兴技术。
