Inada S, Hattori M A, Fujihara N, Morohashi K
Laboratory of Animal Reproduction, Faculty of Agriculture, Kvushu University, Hakozakij Fukuoka, Japan.
Reprod Nutr Dev. 1997;37(1):13-20. doi: 10.1051/rnd:19970102.
An attempt was made to improve gene transfer into chick embryos in order to produce transgenic chickens. The beta-actin-lacZ/MiwZ, a marker gene in transfection reagent, was injected into the blastodisc of either unincubated fertilized eggs (stage X) or eggs induced from the shell gland by treating the hens intravenously with oxytocin or arginine vasotocin (stages IV-VI). All the manipulated embryos were incubated to reach stage XIV, the period at which primordial germ cells (PGCs) migrate from the germinal crescent to the gonadal anlage via the blood stream. MiwZ was detected in the embryos, extraembryonic tissues and blood by the histochemical staining method of beta-galactosidase. The MiwZ DNA was detected in 57% (127/221) of the survival embryos and in 9% (12/127) of the embryonic tissues. The expression was observed mosaically in the epidermis, heart and neural tube. The PGCs in the blood collected from the vitelline artery or dorsal aorta also showed a positive histochemical staining. However, the expression of MiwZ using the soft shelled eggs was more intense in the extraembryonic tissues, although it did not emerge in the embryos. Thus, it is possible to introduce an exogenous gene into the embryonic tissues using incubated fertilized eggs without sacrificing the hens. This technique for successive genetic operations should facilitate the production of transgenic chickens.
为了培育转基因鸡，人们尝试改进向鸡胚的基因转移方法。将转染试剂中的标记基因β-肌动蛋白-乳糖酶基因（MiwZ）注射到未孵化的受精卵（X期）的胚盘，或者通过静脉注射催产素或精氨酸血管加压素诱导母鸡产蛋（IV-VI期）所得到的蛋的胚盘。所有经过操作的胚胎均孵化至XIV期，即原始生殖细胞（PGC）通过血流从生殖新月迁移至性腺原基的时期。通过β-半乳糖苷酶的组织化学染色方法在胚胎、胚外组织和血液中检测到了MiwZ。在57%（127/221）的存活胚胎以及9%（12/127）的胚胎组织中检测到了MiwZ DNA。在表皮、心脏和神经管中观察到了镶嵌性表达。从卵黄动脉或背主动脉采集的血液中的PGC也显示出阳性组织化学染色。然而，使用软壳蛋时，MiwZ在胚外组织中的表达更强，尽管在胚胎中未出现。因此，有可能在不牺牲母鸡的情况下，利用已孵化的受精卵将外源基因导入胚胎组织。这种连续基因操作技术应有助于转基因鸡的培育。
