Analysis of Folate Receptor 4 Function in the Mouse

描述(申请人提供)：叶酸是一种必需的微量营养素，在单碳转移反应中是一种酶辅助因子，如核苷酸合成、DNA甲基化、氨基酸代谢和脂肪合成。叶酸缺乏与出生缺陷和某些类型的癌症的风险有关。哺乳动物细胞已经开发出一种复杂的获取叶酸的机制，包括细胞外叶酸受体，一种跨膜载体，以及一种酶“捕获”策略，以防止叶酸离开细胞。叶酸的本质直观地表明，参与叶酸运输和加工的基因将是在每个细胞中表达的“管家”基因。事实并非如此：在小鼠中，叶酸受体基因在胚胎发育期间非常不同的组织分布中表达。对叶酸受体的功能要求也不同：Folr1突变对胚胎是致命的，但缺乏Folr2基因的小鼠存活。第三种叶酸受体基因存在于小鼠体内，最初命名为folbp3，但与人类FOLR4同源，但缺乏关于该受体的功能信息。这个项目将通过基因敲除的方法来研究Folr4在小鼠中的功能。我们已经产生了一种Folr4基因的‘Folr4’等位基因，它允许我们在所有细胞中或仅在特定组织中使该基因失活。在Folr4胚系缺失的小鼠中，我们将确定对胚胎发育和存活、组织分化以及最终叶酸代谢的影响。除了组织学分析，我们还将测量对其他叶酸受体基因和叶酸代谢途径基因的影响。组织分化的具体焦点将是发育软骨，这是胚胎中高表达Folr4的部位。我们将通过常规的组织学方法和软骨特异性标记的原位杂交结合定量PCR来检测软骨的发育。来自Folr4基因敲除动物和对照的原代软骨细胞将被检测叶酸途径基因的表达。这些实验将揭示叶酸运输受损如何影响整个胚胎和软骨中的细胞生长和功能。Folr4基因敲除小鼠的特征将为未来的实验提供基础，这些实验涉及组织特异性Folr4缺失以及在小鼠基因组中缺失一个以上叶酸受体基因的复合敲除。总之，这些研究将提高我们对叶酸受体基因的基本生物学的理解，并对预防人类出生缺陷产生影响。

DESCRIPTION (provided by applicant): Folate is an essential micronutrient and serves as an enzymatic cofactor in single-carbon transfer reactions, such as nucleotide synthesis, DNA methylation, amino acid metabolism, and lipid synthesis. Folate deficiency has been linked to risk for birth defects and certain types of cancer. Mammalian cells have developed an elaborate mechanism to harvest folate, involving extracellular folate receptors, a transmembrane carrier, and an enzymatic 'trapping' strategy to prevent folate from leaving the cell. The essential nature of folate would intuitively suggest that genes involved in folate transport and processing would be 'housekeeping' genes with expression in every cell. This is not the case: in the mouse, genes for folate receptors are expressed in very distinct tissue distributions during embryonic development. Functional requirements for folate receptors differ as well: A Folr1 mutation is embryonic lethal, yet mice lacking the Folr2 gene survive. A third folate receptor gene exists in the mouse - initially termed folbp3, but homologous to human FOLR4 - but functional information on this receptor is lacking. This project will investigate Folr4 function in the mouse through a gene knockout approach. We have generated mice with a 'floxed' allele of the Folr4 gene that allows us to either inactivate the gene in all cells, or in specific tissues only. In mice with a germline deletion of Folr4, we will determine the effects on embryonic development and survival, tissue differentiation, and, ultimately, folate metabolism. In addition to histological analyses, we will measure the effects on other folate receptor genes and on genes of the Folate metabolic pathway. The specific focus in tissue differentiation will be on developing cartilage, a site of high Folr4 expression in the embryo. We will examine cartilage development by general histological methods and in situ hybridization with cartilage-specific markers together with quantitative PCR. Primary chondrocytes from Folr4 knockout animals and controls will be assayed for expression of Folate pathway genes. These experiments will reveal how impaired folate transport affects cell growth and function in the embryo as a whole and in cartilage. The characterization of Folr4 knockout mice will provide the basis for future experiments involving tissue-specific Folr4 deletions as well as compound knockouts where more than one folate receptor gene is missing from the mouse genome. Together, these studies will improve our understanding of the basic biology of folate receptor genes, with implications for the prevention of human birth defects.