Development of a gene transfer method in utero and a culture method for reconstructed hair germs for study of the process of hair development

为了建立一种方便的胚胎外胚层基因转移方法，在妊娠8.0 ~ 13.5天将含有报告基因Lac-Z或GFP的逆转录病毒注射到小鼠羊膜腔中。应用聚l -赖氨酸后，逆转录病毒在四肢、尾部和背部皮肤上的基因转移效率有所提高。添加聚l -鸟氨酸也有效，但仅在尾皮中有效。当病毒浓度达到10万cfu时，基因转移效率随病毒浓度的增加而提高，但随着病毒注入数量的增加，基因转移效率降低。对于X-gal反应，在妊娠16.0天的全胚中，2% PFA的最佳固定时间为3.5小时。皮肤中可见多种类型的lacz阳性细胞，提示周皮的起源与表皮的起源不同。即使在11周龄小鼠的皮肤中也观察到lacZ和gfp阳性细胞。由于在毛囊中也检测到逆转录病毒，因此子宫内的逆转录病毒感染被认为是将外源基因引入胚胎外胚层并在毛囊中追踪细胞谱系的一种极好的方法。在两步培养的实验中，培养的最重要的一点是毛生殖细胞的温和而完全的分离。用0.25%胰蛋白酶在4℃条件下处理含毛胚皮肤6小时，达到该条件。分离的毛生殖细胞在旋转培养后可以自组装并在重建皮肤中形成聚集体，这一点通过混合gfp标记的毛生殖细胞可以清楚地证明。然而，在漂浮培养过程中，我们未能在它们身上发育出毛囊。因此，在自组装的毛发胚芽和毛发诱导剂（如真皮乳头）之间保持足够的相互作用被认为是重要的。

In order to establish a convenient method for gene transfer into the embryonic ectoderm, retroviruses containing a reporter gene, Lac-Z or GFP, were injected into the amniotic cavity of mice at 8.0 - 13.5 days of gestation. Improvement of gene transfer efficiency by retroviruses was observed in the limb, tail, and back skin when applied with poly-L-lysine. Adding poly-L-ornithine was also effective, but only in the tail skin. Although efficiency of gene transfer enhanced as virus concentration increased up to 100,000 cfu, it reduced if higher number of virus was injected. For X-gal reaction, optimal duration of fixation with 2% PFA was 3.5 hrs for a whole embryo at 16.0 days of gestation. LacZ-positive cells were detected in the skin with various patterns, which suggests that origin of periderm was distinct from one of epidermis. Both lacZ- and GFP-positive cells were observed even in the skin of 11-weeks-old mice. As they were also detected in the hair follicles, retrovirus infection in utero is thought to be an excellent method to introduce exogenous genes into the embryonic ectoderm and to trace cell lineages in the hair follicles.In the experiments of two-step culture, it became clear that the most important point of culture was milder but complete dissociation of hair germ cells. This condition was achieved by treating the hair germ-containing skin with 0.25% trypsin at 4℃ for six hrs. Dissociated hair germ cells could self-assemble and make an aggregate in the reconstructed skin after rotation culture, which was clearly demonstrated by mixing GFP-tagged hair germ cells. However, we failed to develop hair follicles in them during floatation culture. Thus, it is thought to be important to keep an adequate interaction between self-assembled hair germ and hair-inducers such as dermal papillae.