Amendola R, Haendel S, Weier H U, Pallavicini M G
Biomedical Sciences Division, Lawrence Livermore National Laboratory, Livermore, CA 94550.
DNA Cell Biol. 1991 May;10(4):311-7. doi: 10.1089/dna.1991.10.311.
Screening of animals to detect the presence of integrated DNA sequences is an essential component of transgenic mouse generation. Rapid and sensitive detection techniques to facilitate identification of transgenic animals for biological studies or subsequent breeding programs are desirable. Most transgenics are generated on F1 backgrounds, thus determination of the histocompatibility status of neonates provides important diagnostic information for establishing congenic colonies. We describe the application of two assays, in vitro DNA amplification using the polymerase chain reaction (PCR) and fluorescence in situ hybridization with biotinylated DNA probes, to facilitate rapid detection of transgenes and their chromosomal integration patterns in young mice. A noninvasive PCR-based assay to detect the transgene in DNA contained in detergent-extracted hair follicles was developed for rapid screening. A total of 147 mice derived from F2, F3, and F4 generations of C57BL x F1 (globin transgenics) were assayed to determine whether they carried a globin transgene. Characterization of animals by PCR-based amplification of the transgene was compared with that obtained using standard Southern analysis of DNA extracted from tails. Categorization of animals as positive (carrying the transgene) or negative using PCR was performed successfully in the initial assay with 95% of the animals. Fluorescence in situ hybridization with a DNA probe showing homology with a portion of the transgene was performed on metaphase and interphase cells to determine the integration pattern of the transgene. Our data showed that the transgene was integrated in a single chromosome. These techniques should facilitate rapid identification of transgenic animals and characterization of the genomic transgene integration patterns.
筛选动物以检测整合DNA序列的存在是转基因小鼠培育的一个重要组成部分。需要快速且灵敏的检测技术，以便于鉴定用于生物学研究或后续育种计划的转基因动物。大多数转基因动物是在F1背景下产生的，因此确定新生动物的组织相容性状态为建立同源品系提供了重要的诊断信息。我们描述了两种检测方法的应用，即使用聚合酶链反应（PCR）进行体外DNA扩增以及用生物素化DNA探针进行荧光原位杂交，以促进快速检测幼鼠中的转基因及其染色体整合模式。开发了一种基于PCR的非侵入性检测方法，用于检测洗涤剂提取的毛囊中所含DNA中的转基因，以便进行快速筛选。总共对来自C57BL×F1（珠蛋白转基因）F2、F3和F4代的147只小鼠进行检测，以确定它们是否携带珠蛋白转基因。通过基于PCR的转基因扩增对动物进行表征，并与使用从尾巴提取的DNA进行标准Southern分析所获得的结果进行比较。在初始检测中，成功地使用PCR将95%的动物分类为阳性（携带转基因）或阴性。用与转基因一部分具有同源性的DNA探针在中期和间期细胞上进行荧光原位杂交，以确定转基因的整合模式。我们的数据表明，转基因整合在一条单一染色体上。这些技术应有助于快速鉴定转基因动物并表征基因组转基因的整合模式。
