Analysis of Folate Receptor 4 Function in the Mouse

描述（由申请人提供）：叶酸是一种必需的微量营养素，在单碳转移反应中作为酶促辅助因子，如核苷酸合成、DNA甲基化、氨基酸代谢和脂质合成。叶酸缺乏与出生缺陷和某些癌症的风险有关。哺乳动物细胞已经发展出一种复杂的机制来获取叶酸，包括细胞外叶酸受体、跨膜载体和防止叶酸离开细胞的酶“捕获”策略。叶酸的本质直观地表明，参与叶酸运输和加工的基因将是“管家”基因，在每个细胞中都有表达。事实并非如此：在小鼠中，叶酸受体的基因在胚胎发育期间在非常不同的组织分布中表达。叶酸受体的功能需求也不同：Folr1突变是胚胎致命的，而缺乏Folr2基因的小鼠存活。第三种叶酸受体基因存在于小鼠中，最初被称为folbp3，但与人类的FOLR4同源，但缺乏关于该受体的功能信息。本项目将通过基因敲除方法研究Folr4在小鼠中的功能。我们已经培育出了具有Folr4基因“弯曲”等位基因的小鼠，使我们能够在所有细胞中或仅在特定组织中灭活该基因。在种系缺失Folr4的小鼠中，我们将确定其对胚胎发育和存活、组织分化以及最终叶酸代谢的影响。除了组织学分析外，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。组织分化的重点将放在发育中的软骨上，这是胚胎中Folr4高表达的部位。我们将通过一般组织学方法和软骨特异性标记的原位杂交以及定量PCR来检查软骨发育。来自Folr4敲除动物和对照组的原代软骨细胞将被检测叶酸通路基因的表达。这些实验将揭示受损的叶酸运输如何影响整个胚胎和软骨细胞的生长和功能。Folr4敲除小鼠的表征将为未来涉及组织特异性Folr4缺失以及小鼠基因组中多个叶酸受体基因缺失的复合敲除的实验提供基础。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，对预防人类出生缺陷具有重要意义。

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.