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绵羊致死性白综合征携带者的双色高分辨率纤维荧光原位杂交分析

Dual-color high-resolution fiber-FISH analysis on lethal white syndrome carriers in sheep.

作者信息

Pauciullo A, Fleck K, Lühken G, Di Berardino D, Erhardt G

机构信息

Institute of Animal Breeding and Genetics, Justus-Liebig University, DE–35390 Giessen, Germany.

出版信息

Cytogenet Genome Res. 2013;140(1):46-54. doi: 10.1159/000350786. Epub 2013 Apr 26.

Abstract

Molecular defects occurring in the endothelin receptor type-B (EDNRB) gene are known to be associated with pigmentary anomalies and intestinal aganglionosis in humans, rodents and horses. We carried out a cytogenetic investigation in 2 ewes heterozygous for the deletion of the EDNRB gene and in 2 more females as control. The RBA-banding showed that all 4 ewes were karyologically normal. EDNRB gene-specific probes were produced by PCR and cloning. The application of the R-banding and propidium iodide-staining fluorescent in situ hybridization allowed mapping the gene to OAR 10q22 and confirmed the heterozygous status of the ewes investigated for the EDNRB gene deletion. For the fine estimation of the gene length in sheep and for the correct sizing of the chromosomal gap, a dual-color FISH was applied to high-resolution DNA fibers in combination with digital imaging microscopy. The comparison of the DNA fiber barcodes indicated a chromosomal deletion larger than the EDNRB gene itself. The length of the gene, not known for sheep until now, was estimated to be ∼21 kb, whereas the microchromosomal deletion was ∼100 kb. EDNRB is located in a chromosomal region previously shown to be a fragile site. The applied method allowed locating the potential breakpoints, thus permitting further interesting prospective investigations also in the field of the fragile sites in sheep.

摘要

已知内皮素B型受体(EDNRB)基因中出现的分子缺陷与人类、啮齿动物和马的色素异常及肠道神经节细胞缺失有关。我们对2只杂合缺失EDNRB基因的母羊和另外2只作为对照的母羊进行了细胞遗传学研究。RBA显带显示所有4只母羊在核型上均正常。通过PCR和克隆制备了EDNRB基因特异性探针。应用R显带和碘化丙啶染色荧光原位杂交技术将该基因定位到OAR 10q22,并证实了所研究母羊EDNRB基因缺失的杂合状态。为了精确估计绵羊中该基因的长度以及正确确定染色体间隙的大小,将双色荧光原位杂交技术应用于高分辨率DNA纤维,并结合数字成像显微镜。DNA纤维条形码的比较表明染色体缺失大于EDNRB基因本身。此前绵羊的该基因长度未知,估计约为21 kb,而微染色体缺失约为100 kb。EDNRB位于一个先前显示为脆性位点的染色体区域。所应用的方法能够定位潜在的断点,从而也为绵羊脆性位点领域的进一步有趣的前瞻性研究提供了可能。

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