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Cell-based functional genetics of autoimmunity through targeted gene integration

通过靶向基因整合进行基于细胞的自身免疫功能遗传学

基本信息

批准号：
8610877
负责人：
Nunzio Bottini
金额：
$ 22.13万
依托单位：
LA JOLLA INSTITUTE FOR IMMUNOLOGY
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-02-05 至 2016-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8610877
关键词：
Affect Area Autoimmune Diseases Autoimmune Process Autoimmunity Bacterial Artificial Chromosomes Behavioral Research Biological Assay Biomedical Research Cell Line Cell physiology Cells Clinical Research Clone Cells Cloning Collection Complex Data Detection Development Diagnosis Disease Distant Exploratory/Developmental Grant Fostering Funding Gene Combinations Gene Expression Gene Targeting Generations Genes Genetic Genetic Polymorphism Genetic Variation Genome Genomic DNA Genomics Genotype Goals Gold Grant Hand Haplotypes Human Human Cell Line IL2RA gene Individual Interleukin-2 Laboratories Lead Learning Left Length Linkage Disequilibrium Location Maps Methodology Methods Modeling Molecular Molecular and Cellular Biology Nature PTPN22 gene Parents Patients Problem Solving Proteins RNA Splicing Regulator Genes Reporter Risk Series Signal Transduction Single Nucleotide Polymorphism Speed Spliced Genes System T-Lymphocyte Techniques Technology Transfection Transgenic Organisms Variant Work base gene interaction genetic variant genome wide association study high risk human disease improved innovation novel novel strategies prototype receptor research study tool transgene expression

项目摘要

In the last five years, dozens of new genes have been identified that increase or decrease the risk of autoimmune diseases. Many laboratories are now trying to determine how certain autoimmune-predisposing or -protective haplotypes (i.e. combinations of gene polymorphisms) affect the expression, splicing, and function of the encoded protein. However, the current approach, based on studying primary cells from individuals carrying different haplotypes, often gives unclear answers. This grant will try to validate a novel approach to functional genetics of autoimmunity, which is complementary to that for primary cells. Our strategy is to assess functional differences between autoimmune-predisposing and protective genetic variations by directly studying full-length gene haplotypes transfected in human cells. The full-length gene will be carried on a bacterial artificial chromosome (BAC) and manipulated before transfection in order to carry the desired variations. The result will be a series of cell clones that carry different full-length gene haplotypes. The clones will then be subjected to a variety of functional studies. We already have a prototype of the system in-hand, and here we apply for the funding needed to validate our approach and optimize it for large-scale application. Thus we will focus on two well-known autoimmunity genes relevant for T cell function, PTPN22 and IL2RA, and use Jurkat T cells as a model cell line. In Aim 1 we will use our prototype assay to assess the functional effect of gene variations in PTPN22 and IL2RA. In Aim 2 we will experiment with long-PCR-based approaches in the attempt to achieve cloning of haplotypes into BACs directly from patient genomic DNA. This will eliminate the need for gene manipulation in order to obtain the desired haplotypes. Our approach is novel and simple, and does not require collection of primary cells. Once validated and optimized, it can be applied to all autoimmunity genes using a variety of cell lines, and even broadly applied to functional genetics studies of complex human diseases.

在过去的五年里，已经发现了几十种新的基因，它们可以增加或减少自身免疫性疾病的风险。许多实验室现在正试图确定某些自身免疫易感性或保护性单倍型（即基因多态性的组合）如何影响编码蛋白的表达、剪接和功能。然而，目前的方法，基于研究来自携带不同单倍型个体的原代细胞，往往给出不明确的答案。这项拨款将试图验证一种新的方法，以功能遗传学的自身免疫，这是补充原代细胞。我们的策略是通过直接研究转染人细胞的全长基因单倍型来评估自身免疫易感性和保护性遗传变异之间的功能差异。全长基因将携带在细菌人工染色体（BAC）上，并在转染前进行操作，以携带所需的变异。结果将是一系列携带不同全长基因单倍型的细胞克隆。然后，克隆人将接受各种功能研究。我们已经有了一个系统的原型，在这里，我们申请所需的资金来验证我们的方法，并为大规模应用优化它。因此，我们将重点关注与T细胞功能相关的两个众所周知的自身免疫基因PTPN22和IL2RA，并使用Jurkat T细胞作为模型细胞系。在Aim 1中，我们将使用我们的原型分析来评估PTPN22和IL2RA基因变异的功能影响。在目标2中，我们将实验基于长链聚合酶链反应的方法，试图将单倍型直接从患者基因组DNA克隆到bac中。这将消除为了获得所需的单倍型而进行基因操作的需要。我们的方法新颖而简单，而且不需要收集原代细胞。一旦验证和优化，它可以应用于使用各种细胞系的所有自身免疫基因，甚至广泛应用于复杂人类疾病的功能遗传学研究。