2002 Fiscal Year Final Research Report Summary
Development of a gene transfer method in utero and a culture method for reconstructed hair germs for study of the process of hair development
| Project/Area Number |
13670887
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (C)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 一般 |
|---|
| Research Field |
Dermatology
|
|---|
| Research Institution | Shimane University |
|---|
Principal Investigator |
MATSUZAKI Takashi Shimane Univ., Fac. Life & Environmental Science, Assistant prof., 生物資源科学部, 講師 (90241249)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
IHARA Setsunosuke Shimane Univ., Fac. Life & Environmental Science, Professor, 生物資源科学部, 教授 (90101295)
|
|---|
| Project Period (FY) |
2001 – 2002
|
| Keywords | retrovirus / gene transfer in utero / keratinocyte / hair follicle / cell differentiation / reconstraction |
|---|
| Research Abstract |
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.
|
Research Products
(12 results)
-
-
-
-
-
-
-
[Publications] K,Morioka., K,Sato-Kusubata., S,Kawashima., T,Ueno., E,Kominami., H,Sakuraba., S,Ihara.: "Localization of cathepsins B, D L, LAMP-1 and m-calpain in developing hair follicles."Acta Histochem. Cytocham. 34. 337-347 (2001)
Description
「研究成果報告書概要(欧文)」より
-
-
-
-
-
