FIRST HSCS DURING MOUSE ONTOGENY

小鼠个体发育期间的首次 HSCS

基本信息

批准号：
2906242
负责人：
ELAINE A DZIERZAK
金额：
$ 11.45万
依托单位：
ERASMUS UNIVERSITY OF ROTTERDAM
依托单位国家：
美国
项目类别：
财政年份：
1998
资助国家：
美国
起止时间：
1998-06-01 至 2001-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2906242
关键词：
blood circulation cytokine receptors embryo /fetus cell /tissue embryogenesis flow cytometry gene expression genetic markers genetic promoter element genetically modified animals hematopoiesis hematopoietic stem cells immunocytochemistry in situ hybridization laboratory mouse mutant organ culture polymerase chain reaction receptor expression transforming growth factors

blood circulation cytokine receptors embryo /fetus cell /tissue embryogenesis flow cytometry gene expression genetic markers genetic promoter element genetically modified animals hematopoiesis hematopoietic stem cells immunocytochemistry in situ hybridization laboratory mouse mutant organ culture polymerase chain reaction receptor expression transforming growth factors

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项目摘要

DESCRIPTION: The P.I. intends to identify the developmental origin of the hematopoietic stem cell which can repopulate the hematopoietic system of adult mice. The proposal is based on solid preliminary observations made by the P.I. that the earliest stage and site of repopulating stem cells in the mouse is in the AGM region of day 10 mouse embryos. repopulating cells appear in the yolk sac and fetal liver subsequent to day 10. Since circulation is well established by day 10, hypotheses holding that stem cells arise de novo in the AGM region and/or seed there and elsewhere by entering the circulation are viable. This proposal tests that hypothesis. Aim 1 will make use of organ culture to identify the origin of the earliest repopulating stem cells. This use of organ culture removes the complications of circulation from the analysis, and preliminary experiments show that organs can be cultured from 8 to 11 day embryos. Yolk sac, liver primordium, and the AGM region will be examined and limiting dilution will be used to estimate the number of CFU-S and repopulating stem cells. Verification of multilineage repopulation will be provided by genetic markers and if necessary retroviral marking. Both procedures have been used in the past by the P.I. for related studies. Once the origin of repopulating stem cells is established, the P.I. proposes to investigate the emergence of repopulating stem cells in transgenic mice which are deficient in GATA-2, and 3. These animals have defects in definitive hematopoiesis. Finally, the P.I. will examine the cell surface characteristics of AGM stem cells. These studies will be aided by transgenic mice which express lac Z under the control of the Sca-1 promoter, aiding in the identification and further characterization of these cells. In aim 2, the P.I. will examine the microenvironment in the yolk sac for its ability to support AGM derived HSC. The use of organ culture will allow the isolation of HSC free yolk sac cells on which various normal and mutant HSCs will be grown. The pattern of cytokine and cytokine receptor gene expression, as well as TGFb family members will be examined in stromal cells using RT-PCR. This information will be compared to immunohistochemistry of embryo tissues which has been done previously or will be done in parallel with the RT-PCR analysis.

描述：P.I。打算确定造血干细胞，可以重新填充造血系统成年小鼠。该提案基于坚实的初步观察。 P.I.最早的阶段和位点，在小鼠处于第10天小鼠胚胎的AGM区域。重新流动细胞出现在第10天之后的蛋黄和胎儿肝脏中。在第10天之前，循环已建立得很好，假设持有茎细胞在AGM地区和/或在那里和其他地方出现。进入循环是可行的。该建议检验了该假设。 AIM 1将利用器官文化来确定最早的起源重新流动干细胞。器官文化的这种使用可以消除分析和初步实验的循环并发症证明器官可以从8到11天的胚胎进行培养。蛋黄囊，肝脏原始和AGM区域将进行检查，并将限制稀释用于估计CFU-S的数量和重新填充的干细胞的数量。遗传将提供多列元重生的验证标记和必要的逆转录病毒标记。两种程序均已使用过去由P.I.用于相关研究。一旦起源于建立了重生干细胞，P.I。建议调查不足的转基因小鼠中重室干细胞的出现在GATA-2和3中。这些动物在确定的造血中存在缺陷。最后，P.I.将检查AGM茎的细胞表面特征细胞。这些研究将由表达LAC Z的转基因小鼠帮助在SCA-1启动子的控制下，有助于识别和这些细胞的进一步表征。在AIM 2中，P.I.将检查蛋黄囊中的微环境是否有支持AGM派生的HSC的能力。器官文化的使用将允许隔离HSC游离蛋黄囊细胞，在其上有各种正常和突变的HSC 会成长。细胞因子和细胞因子受体基因的模式表达以及TGFB家族成员将在基质细胞中检查使用RT-PCR。该信息将与免疫组织化学进行比较以前已经完成或将并行完成的胚胎组织通过RT-PCR分析。