Full karyotype single cell interphase analysis

全核型单细胞间期分析

• 批准号: 6644520
• 负责人: SANTIAGO MUNNE
• 金额: $ 11.93万
• 依托单位: REPROGENETICS, INC.
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-28 至 2004-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6644520
• 关键词: aneuploidy; biotechnology; chromosomes; diagnosis design /evaluation; egg /ovum; fluorescent in situ hybridization; genetic disorder diagnosis; genetic screening; human tissue; karyotype; nucleic acid probes; technology /technique development

DESCRIPTION (provided by applicant): Early human development depends on the correct temporal and spatial expression of thousands of genes. The presence of an extra chromosome or a chromosome missing in gametes typically leads to failed fertilization or spontaneous abortions, resulting in phenotypes called 'reduced fertility'. Reduced fertility or infertility may have one of many reasons, but is largely a consequence of advanced age, presence of a structural chromosome abnormality such as a translocation or for familial reasons. Many patients suffering from reduced fertility can now be helped to get the babies they have been hoping for though a combination of in vitro fertilization, which increases the number of embryos available for replacement, and 'Preimplantation Genetic Diagnosis (PGD)', which selects those embryos that are chromosomically normal for replacement. The clinical approach is termed 'Preimplantation Genetic Diagnosis (PGD)' of aneuploidy. PGD is based on interphase cell analysis using fluorescence in situ hybridization (FISH). This approach is sensitive to time constraints and the number of cells available (i.e., 1 or 2). Similarly, for neoplastic analysis or fetal peripheral blood sometimes it is necessary to analyze very small samples of tissue, which will require also interphase analysis of chromosomes. Alternative techniques, such as SKY or CGH, cannot be used effectively for PGD because require either metaphase spreads, which cannot be reliable obtained from one single cell, or need too many- days of hybridization, which is incompatible with regular in vitro fertilization. However, current FISH technology can only detect a limited number of chromosome abnormalities in interphase cells. Thus we propose to combine a novel FISH protocol with Spectral Imaging (SIm) detection to determine the exact number and types of all chromosomes, by using up to 8 uniquely labeled, chromosome-specific DNA probes, in three sequential rounds of hybridization. Our previous experience with FISH suggests that cells can safely go through 3 rounds of FISH without losing much specificity. Thus combining three rounds of hybridizations with 8 different sets of probes per hybridization will allow us analyze all 24 types of chromosomes in a single cell. In a previous SBIR grant (1R43HD3501001A 1) we demonstrated that Sim could work in polar bodies biopsied from oocytes, but that interphase FISH-Sim will solve problems related to chromosome overlap and nuclear condensation that affect chromosome painting in metaphase stage nuclei. In phase-I we propose to develop two of the three sets of probes, and to test that these probes will work with 90% efficiency after three rounds of hybridization in single polar bodies.

描述（由申请人提供）：人类早期发育取决于数千个基因的正确时空表达。配子中存在额外的染色体或染色体缺失通常会导致受精失败或自然流产，导致称为“生育力降低”的表型。生育力下降或不育可能有许多原因之一，但主要是高龄，结构染色体异常如易位或家族原因的结果。 许多患有生育能力下降的患者现在可以通过体外受精和“植入前遗传诊断”的结合来帮助他们获得他们一直希望的婴儿，体外受精增加了可供替换的胚胎数量，“植入前遗传诊断”选择了染色体正常的胚胎进行替换。 临床方法被称为非整倍体的“植入前遗传学诊断（PGD）”。PGD基于使用荧光原位杂交（FISH）的间期细胞分析。该方法对时间约束和可用小区的数量（即，1或2）。类似地，对于肿瘤分析或胎儿外周血，有时需要分析非常小的组织样品，这也需要染色体的间期分析。替代技术（例如SKY或CGH）无法有效用于PGD，因为要么需要中期扩散（无法从一个单细胞中可靠地获得），要么需要太多天的杂交，这与常规体外受精不相容。然而，目前的FISH技术只能检测到有限数量的间期细胞染色体异常。因此，我们建议联合收割机一种新的FISH协议与光谱成像（SIM）检测，以确定所有染色体的确切数量和类型，通过使用多达8个独特的标记，染色体特异性DNA探针，在三轮连续杂交。我们以前的FISH经验表明，细胞可以安全地通过3轮FISH，而不会失去太多的特异性。因此，将三轮杂交与每次杂交的8组不同探针组合将允许我们分析单个细胞中的所有24种类型的染色体。在以前的SBIR资助（1 R43 HD 3501001 A 1），我们证明，SIM可以在极体活组织检查从卵母细胞，但间期FISH-SIM将解决染色体重叠和核凝聚的问题，影响染色体绘画中期阶段的细胞核。在第一阶段，我们建议开发三套探针中的两套，并测试这些探针在单极体中三轮杂交后将以90%的效率工作。