CHROMOSOME MAPPING IN DOMESTIC ANIMALS BY IN SITU HYBRIDIZATION

本研究旨在建立家畜染色体上基因定位的原位杂交方法。初步以~3H标记的卵磷脂-胆固醇酰基转移酶DNA(16q22)为探针，对人染色体进行原位杂交。然后，为了建立牛染色体原位杂交的方法，以~3H标记的牛低密度脂蛋白受体DNA为探针。然而，由于该DNA可能不适合作为原位杂交的探针，因此无法确定该基因的位置。尝试使用生物素标记的α卫星DNA探针进行荧光原位杂交(FISH)以定位人类染色体上的基因。经抗生物素-异硫氰酸荧光素处理和碘化丙啶反染后，在染色体上检测到荧光信号。他们已经显示了阿尔法卫星DNA的位置。建立的FISH方法被应用于转基因大鼠染色体上的转基因定位。YS新技术研究所将小鼠乳清酸性蛋白(m-WAP)注入大鼠原核卵子，获得转基因大鼠外周血淋巴细胞中期染色体铺展。染色体在FISH前用Tripsin技术进行G显带，通过核型分析，将转基因(m-WAP)定位在6号染色体的Q14位，为家畜基因定位奠定了基础。

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.