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

开发子宫内基因转移方法和重建毛胚培养方法以研究毛发发育过程

基本信息

批准号：
13670887
负责人：
MATSUZAKI Takashi
金额：
$ 2.05万
依托单位：
Shimane University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13670887/
关键词：
retrovirus gene transfer in utero keratinocyte hair follicle cell differentiation reconstraction

项目摘要

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.

为了建立一种简便的方法将基因转移到胚胎外胚层中，将含有报告基因Lac-Z或GFP的逆转录病毒注射到妊娠8.0 - 13.5天的小鼠的羊膜腔中。在四肢、尾部和背部皮肤中观察到应用聚-L-赖氨酸时逆转录病毒的基因转移效率的改善。添加聚-L-鸟氨酸也有效，但仅在尾部皮肤中。尽管当病毒浓度增加到100，000cfu时基因转移效率增强，但如果注射更高数量的病毒，则基因转移效率降低。对于X-gal反应，用2%PFA固定的最佳持续时间为3.5小时，对于妊娠16.0天的整个胚胎。LacZ阳性细胞在皮肤中以不同的方式检测到，这表明周皮的起源与表皮不同。即使在11周龄小鼠的皮肤中也观察到lacZ和GFP阳性细胞。由于在毛囊中也能检测到，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中细胞谱系的一种很好的方法。在两步培养实验中，很明显培养的最重要的一点是温和但完全分离的毛生殖细胞。通过在4 ℃下用0.25%胰蛋白酶处理含毛胚的皮肤6小时来实现该条件。旋转培养后，游离的毛生殖细胞可以在重建皮肤中自组装并形成聚集体，混合GFP标记的毛生殖细胞可以清楚地证明这一点。然而，在漂浮培养过程中，我们未能在他们身上发育毛囊。因此，保持自组装的毛胚和毛发诱导物如真皮乳头之间的充分相互作用被认为是重要的。