Cell lineage analyses for histogenesis and diversification of the telencephalon

端脑组织发生和多样化的细胞谱系分析

基本信息

批准号：
18570203
负责人：
SHIMAMURA Kenji
金额：
$ 2.63万
依托单位：
Kumamoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

项目摘要

It is believed that cellular diversity found in the various regions of the brain is primarily derived from regionally distinct properties of the neural progenitors. To elucidate the extent to which the regional differences found in the mature brain structures depend upon the early neuroepithelial regionality, we decided to conduct a fate map study based upon gene expression, using Cre/loxP recombination system. We have generated transgenic mouse lines in which a tamoxifen-inducible form of Cre recombinase (CreERT2) was driven under the control of Six3 locus, a gene expressed in the anterior forebrain primordium in a temporary dynamic pattern. We took a knock-in strategy to recapitulate precisely the expression of endogenous Six3 gene. Three neomycin-resistant ES clones as well as mouse lines derived from those clones were established. A Cre-reporter line Rosa26R was crossed with the heterozygous of Six3-CreERT2neo and tamoxifen was delivered to the mother which enables Cre to excise the floxed stop cassette that transcriptionally activates lacZ reporter. While the LacZ-expressing cells were to be detected as the descendant of the progenitors that expressed Six3 at a given time, we found very few X-gal cells within the Six3-expressing domains of the forebrain which were variable among the specimens and within them sometimes (laterally asymmetric) due to very low level of Cre-expression. It was essentially the same, even after the FRT-flanked pgk-neo-pA cassette was excised by crossing with the CAGGS-Flpw mice, although the number of LacZ-positive cells was significantly increased. We thus concluded that these lines are not to be used for the stage-specific lineage analysis of the Six3-expressing domains. Although these mice turned out to be inappropriate for our original purpose, recombination in small subsets of cells in those domains could potentially be useful for detailed analysis (e. g. mosaic) as a Cre-driver line for the conditional knock out studies.

据认为，在大脑的各个区域中发现的细胞多样性主要来自神经祖细胞的区域特性。为了阐明在成熟的大脑结构中发现的区域差异取决于早期的神经上皮区域，我们决定使用CRE/LOXP重组系统基于基因表达进行命运图研究。我们已经产生了转基因小鼠系，其中他莫昔芬可诱导的Cre重组酶（Creert2）的形式是在六号基因座的控制下驱动的，这是一个以临时动力学模式在前前脑原始基因中表达的基因。我们采取了一种敲门策略，以精确地概括了内源性六个基因的表达。建立了三个耐新霉素的ES克隆以及从这些克隆得出的小鼠线。将Cre-Reporter系列Rosa26R与六个-Creert2neo的杂合子交叉，并将其莫昔芬交付给母亲，使Cre可以切除转录激活Lacz Reporter的Floxed Stop Cassette。尽管在给定时间表达了六3的祖细胞的后代，但在给定时间表达了63个，我们发现前脑的X-Gal细胞很少，由于CRE-表达非常低的情况，我们有时由于有时在标本中的六个表达域（横向不对称）。即使在通过与CAGGS-FLPW小鼠交叉切除FRT频率的PGK-NEO-PA盒子，尽管LACZ阳性细胞的数量显着增加，但它基本相同。因此，我们得出的结论是，这些线不适用于对六个表达域的阶段特定谱系分析。尽管事实证明这些小鼠不适合我们的最初目的，但这些域中小细胞的重组中的重组可能对于详细分析（例如Mosaic）作为条件敲除研究的CRE驱动器线可能有用。