Full karyotype single cell interphase analysis

描述(申请人提供)：早期人类发育依赖于数千个基因的正确时间和空间表达。配子中存在额外的染色体或缺失的染色体通常会导致受精失败或自然流产，从而导致被称为“生育能力降低”的表型。生育力下降或不孕不育可能有许多原因之一，但主要是由于高龄、染色体结构异常(如易位)或家庭原因造成的。 许多生育力低下的患者现在可以通过体外受精和植入前遗传诊断(PGD)相结合的方式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(PGD)选择了染色体正常的胚胎进行替换。 这种临床方法被称为非整倍体的植入前遗传学诊断(PGD)。PGD是基于使用荧光原位杂交(FISH)的间期细胞分析。这种方法对时间限制和可用的单元格数量(即1或2个)很敏感。同样，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本，这也需要对染色体进行间期分析。替代技术，如Sky或CGH，不能有效地用于PGD，因为要么需要中期铺展，这不能从一个单一细胞获得可靠的结果，要么需要太多天的杂交，这与常规的体外受精不相容。然而，目前的FISH技术只能检测到间期细胞中有限数量的染色体异常。因此，我们建议将一种新的FISH方法与光谱成像(SIM)检测相结合，通过在三轮连续的杂交中使用多达8个唯一标记的、染色体特定的DNA探针来确定所有染色体的确切数量和类型。我们之前对FISH的经验表明，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，结合三轮杂交和每一次杂交8组不同的探针，我们将能够在单个细胞中分析所有24种类型的染色体。在之前的SBIR拨款(1R43HD3501001A1)中，我们证明了SIM可以在从卵母细胞活检的极体中工作，但间期FISH-SIM将解决与染色体重叠和核凝聚相关的问题，这些问题影响中期细胞核的染色体绘制。在第一阶段，我们建议开发三组探针中的两组，并测试这些探针在单极体中进行三轮杂交后将以90%的效率工作。

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.