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

为了建立一种简便的基因转移到胚胎外胚层的方法，将含有报告基因Lac-Z或GFP的逆转录病毒注射到孕8.0-13.5天的小鼠羊膜内。当应用多聚L赖氨酸时，可以观察到逆转录病毒提高了肢体、尾部和背部皮肤的基因转移效率。添加聚L鸟氨酸也有效，但仅在尾部皮肤有效。虽然基因转移效率随着病毒浓度的增加而提高，但随着病毒数量的增加，基因转移效率降低。对于X-Gal反应，2%PFA固定整个胚胎的最佳时间为16.0d的3.5h。在皮肤中检测到不同形态的LacZ阳性细胞，表明周皮的起源与表皮的起源不同。甚至在11周大的小鼠皮肤中也能观察到LacZ和GFP阳性细胞。由于在毛囊中也检测到逆转录病毒，因此宫内逆转录病毒感染被认为是将外源基因导入胚胎外胚层和追踪毛囊细胞谱系的一种很好的方法。在两步培养实验中，很明显，最重要的是培养的重点是较温和但完全分离的毛囊生殖细胞。该条件是用0.25%胰酶在4℃下处理毛发胚芽皮肤6小时。分离的毛细胞在旋转培养后可以在重建皮肤中自组装并形成聚集体，这一点通过混合GFP标记的毛细胞得到了清楚的证明。然而，在漂浮培养过程中，我们未能在其中发育毛囊。因此，在自组装毛发细菌和毛乳头等毛发诱导剂之间保持适当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.