Chromosomal mapping and zygosity check of transgenes based on flanking genome sequences determined by genomic walking
Chromosomal mapping and zygosity check of transgenes based on flanking genome sequences determined by genomic walking
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DOI：
10.1538/expanim.53.103
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发表时间：
2004-04-01
影响因子：
2.4
通讯作者：
Suzuki, O
中科院分区：
文献类型：
--
作者：
Noguchi, A;Takekawa, N;Suzuki, O
转基因可以通过转基因表达或通过所谓的位置效应影响转基因小鼠。使用最近建立的小鼠基因组数据库可以将 DNA 序列定位在染色体中。在这项研究中，我们描述了一种染色体作图方法，该方法使用基因组行走技术结合小鼠基因组数据库搜索来分析转基因侧翼的基因组序列。从含有基于 pCAGGS 的转基因的两个转基因小鼠系收集基因组 DNA，并为每个小鼠系构建了接头连接的酶限制性基因组文库。通过使用转基因特异性引物和接头引物对基因组文库进行 PCR 扩增获得的扩增子进行测序来确定侧翼序列。使用转基因的侧翼序列对Ensembl基因组数据库进行BLAST搜索来定位转基因的插入位置，并将两个转基因小鼠品系的转基因定位到11号和3号染色体上。此外，使用侧翼序列信息构建侧翼引物以进行接合性检查。动物的接合性（纯合转基因、半合转基因和非转基因）可以通过使用侧翼引物的PCR分析中的差异条带形成来鉴定。尽管需要考虑转基因整合的模式和侧翼序列的特异性，但这些方法对于转基因动物的遗传质量控制应该是有用的。
Transgenes can affect transgenic mice via transgene expression or via the so-called positional effect. DNA sequences can be localized in chromosomes using recently established mouse genomic databases. In this study, we describe a chromosomal mapping method that uses the genomic walking technique to analyze genomic sequences that flank transgenes, in combination with mouse genome database searches. Genomic DNA was collected from two transgenic mouse lines harboring pCAGGS-based transgenes, and adaptor-ligated, enzyme restricted genomic libraries for each mouse line were constructed. Flanking sequences were determined by sequencing amplicons obtained by PCR amplification of genomic libraries with transgene-specific and adaptor primers. The insertion positions of the transgenes were located by BLAST searches of the Ensembl genome database using the flanking sequences of the transgenes, and the transgenes of the two transgenic mouse lines were mapped onto chromosomes 11 and 3. In addition, flanking sequence information was used to construct flanking primers for a zygosity check. The zygosity (homozygous transgenic, hemizygous transgenic and non-transgenic) of animals could be identified by differential band formation in PCR analyses with the flanking primers. These methods should prove useful for genetic quality control of transgenic animals, even though the mode of transgene integration and the specificity of flanking sequences needs to be taken into account.