Endocrine cell induction during pancreas

胰腺发育期间从祖细胞形成胰岛需要Delta/Notch信号通路的协调激活。尽管这一途径与维持多能性和支持胰腺祖细胞的细胞命运决定有关，但对其了解甚少。为了更好地了解胰腺发育过程中发生的这些和其他事件，重要的是能够容易地检查发育中的小鼠胰腺内功能获得和功能丧失突变的影响。在可能的情况下，这些突变是有条件活性的或无活性的也是有用的。因此，该项目的重点是在小鼠ES细胞中使用和进一步开发盒交换技术。这种策略加速了ES细胞中高度确定的基因突变的产生，然后可以用于制造新的小鼠或作为学习如何将ES细胞分化为胰腺或β细胞命运的工具。将要开发的试剂以及将要进行的实验有望加速对胰腺内分泌细胞产生的分子事件的理解。在目的1中，我们将使用在Ptf 1a基因座控制下表达黄色荧光蛋白的小鼠来探索胰腺祖细胞的生长和分化能力。对于目标2，我们将探索 通过在Ptf 1a基因座的控制下表达三种不同的边缘基因的Delta/Notch信号传导。在目标3中，我们将评估Insm 1的整体敲除的效果，同时还使用改进的策略产生新的loxed盒受体等位基因。对于目标4，我们将产生和测试新的小鼠ES细胞系，这将有助于学习如何引导这些细胞向胰腺β细胞命运分化。最后，在目标5中，我们将建立一种新的盒式交换系统，该系统利用phiC 31整合酶将基因快速插入ROSA 26基因座，并随后通过Cre以位点特异性和时间特异性方式诱导基因。

The formation of Islets of Langerhans from progenitor cells during pancreas development requires the coordinated activation of the Delta/Notch signaling pathway. This pathway, although it has been implicated in both maintaining multipotency and supporting cell fate decisions of pancreatic progenitor cells, is poorly understood. In order to better understand these and other events that occur during pancreas development, it is important to be able to easily examine the effects, within the developing mouse pancreas, of both gain-of-function and loss-of-function mutations. When possible, it is also useful for these mutations to be conditionally active or inactive. Thus, this project is focused on both the use and further development of cassette exchange technologies in mouse ES cells. This strategy accelerates the production of highly defined gene mutations in ES cells, which can then be used either to make new mice or as tools for learning how to direct the differentiation of ES cells towards a pancreatic or beta cell fate. The reagents that will be developed, as well as the experiments that will be performed, promise to accelerate progress towards an understanding of the molecular events that give rise to endocrine cells within the pancreas. In Aim 1 we will explore the growth and differentiation capacity of pancreatic progenitor cells using mice in which yellow fluorescent protein is expressed under control of the Ptf1a gene locus. For Aim 2 we will explore alterations in Delta/Notch signaling by expressing three different fringe genes under control of the Ptf1a gene locus. In Aim 3 we will assess the effects of a global knock-out of Insm1 while also generating a new loxed cassette acceptor allele using an improved strategy. For Aim 4 we will generate and test new mouse ES cell lines that will facilitate learning how to direct the differentiation of these cells towards a pancreatic beta cell fate. Lastly, in Aim 5 we will establish a new cassette exchange system that makes use of phiC31 integrase to allow rapid insertion of genes into the ROSA26 locus and their subsequent induction in a site- and temporal-specific manner by Cre.