Chromosomal mapping and zygosity check of transgenes based on flanking genome sequences determined by genomic walking

Akira Noguchi, Naho Takekawa, Thorbjorg Einarsdottir, Minako Koura, Yoko Noguchi, Kaoru Takano, Yoshie Yamamoto, Junichiro Matsuda, Osamu Suzuki*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

12 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)103-111
Number of pages9
JournalExperimental Animals
Volume53
Issue number2
DOIs
Publication statusPublished - 2004

Other keywords

  • Chromosomal mapping
  • Flanking primers
  • Genomic walking
  • Zygosity check

Fingerprint

Dive into the research topics of 'Chromosomal mapping and zygosity check of transgenes based on flanking genome sequences determined by genomic walking'. Together they form a unique fingerprint.

Cite this