CHROMOSOME MAPPING IN DOMESTIC ANIMALS BY IN SITU HYBRIDIZATION

通过原位杂交对家养动物进行染色体定位

• 批准号: 03660274
• 负责人: YOSHIZAWA Midori
• 金额: $ 1.34万
• 依托单位: UTSUNOMIYA UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1991
• 资助国家: 日本
• 起止时间: 1991 至 1993
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-03660274/
• 关键词: CATTLE; CHROMOSOME; LOCALIZATION; MAPPING; TRANSGENIC RAT; IN SITU HYBRIDIZATION; FLUORESCENCE IN SITU HYBRIDIZATION; 蛍光in situ hybridization; 高精度分染; ビオチン; DNA; 遺伝子; 染色体地図; Gーバンド

The present study aimed to establish a method of localization of genes in chromosomes of domestic animals by in situ hybridization.Preliminarily, ^3H-labeled LCAT (lecithin-cholesterol acyltransferase)DNA,whose locus has been known(No.16q22), was used for in situ hybridization of human chromosomes as a probe. Then, in order to establish a method of in situ hybridization in bovine chromosomes, ^3H-labeled bovine LDL (low-density lipoprotein)receptor DNA was used as a probe. However, the locus was not determined because this DNA might be not suitable for in situ hybridization as the probe.Fluorescence in site hybridization(FISH) was attempted to localize a gene on human chromosomes using alpha-satellite DNA probe labeled with biotin. After treatment with avidin-fluorescein isothiocyanate and counterstain with propidium iodide, fluorescent signals were detected on chromosomes. They have shown the loci of alpha-satellite DNA.The established FISH method was applied to localization of a transgene on chromosomes of transgenic rats. Metaphase chromosome spreads were obtained from peripheral blood lymphocyte cultures of transgenic rats, which were produced by insertion of mouse whey acidic protein(m-WAP) into pronuclear rat eggs in YS New Techonology Institute. Chromosomes were G-banded by tripsin technique before FISH.The transgene (m-WAP)was localized at q14 site of no.6 chromosome by karyotype analysis.It is considered that the present study contributes to the gene mapping in domestic animals.

为了建立家畜染色体上基因的原位杂交定位方法，首先，以16 q22位点已知的卵磷脂胆固醇酰基转移酶（LCAT）DNA为探针，进行了人染色体的原位杂交。然后，为了建立牛染色体原位杂交的方法，我们用^3H标记的牛LDL（低密度脂蛋白）受体DNA作为探针。然而，由于该DNA可能不适合作为探针进行原位杂交，因此未能确定该基因座。利用生物素标记的α-卫星DNA探针进行荧光原位杂交（Fluorescence in site hybridization，FISH），试图对人类染色体上的基因进行定位。经抗生物素蛋白-异硫氰酸荧光素处理和碘化丙啶复染后，在染色体上检测到荧光信号。将建立的FISH方法应用于转基因大鼠的染色体定位。从YS新技术研究所的小鼠乳清酸性蛋白（m-WAP）插入原核大鼠卵细胞制备的转基因大鼠外周血淋巴细胞培养物中获得中期染色体铺展。FISH前用胰蛋白酶技术对染色体进行G显带，核型分析将转基因（m-WAP）定位于第6号染色体q14位点，认为本研究有助于家畜基因定位。