(R3364) Transgene Mapping and Genotyping Protocol Design in a Transgenic Mouse Strain by Short-Read Whole-Genome Sequencing

Osamu Suzuki (National Institutes of Biomedical Innovation, Health and Nutrition)| Minako Koura (National Institutes of Biomedical Innovation, Health and Nutrition)| Kozue Uchio (National Institutes of Biomedical Innovation, Health and Nutrition)| Mitsuho Sasaki (National Institutes of Biomedical Innovation, Health and Nutrition)| Yuko Doi (National Institutes of Biomedical Innovation, Health and Nutrition)| Liang Bing (National Institutes of Biomedical Innovation, Health and Nutrition)

[Aim] When creating transgenic mice, multiple lines with the same transgene are often created. For example, our laboratory has three lines of hAce2 transgenic mice (hAce2-Tg). For one of them (hAce2-Tg#16), we mapped the transgene using short-read whole-genome sequencing and designed genotyping PCR to identify the line. [Methods] We extracted genomic DNA from kidneys of hAce2-Tg#16. Using PCR-free libraries prepared with the genomic DNA, paired-end 150-bp reads were performed by DNBSEQG-400 sequencer (MGI Tech) (outsourced). In order to find the junction between the genome and the transgene, we created two search programs: one to search for matches between the transgene and each read and its complementary sequence from the front (5' side, head-match), and the other to search for matches from the back (3' side, tail-match). We selected reads with a match length of 5 to 145 bases. After the search, we selected reads containing the mouse genome sequence in the non-matching part by BLAST search of the Ensembl database. Based on this sequence information, we designed primers for genotyping PCR based on the flanking primer method. All animal experiments were conducted in accordance with the guidelines for animal experiments of the National Institutes of Biomedical Innovation, Health, and Nutrition, Osaka, Japan (authorization number: DS22-55). [Results and Discussion] In a total of 882,577,032 reads (total read length: about 132 GBase), the total numbers of head-matching and tail-matching reads were 1,498 and 3,060, respectively. Among them, eight reads in the head-matching reads had the same genomic sequence. Its genomic location was in the intron of the Nrxn3 gene on chromosome 12. The transgene was truncated by 82 bases from the 3' end. On the other hand, none of the tail-matching reads contained the neighboring genomic sequence. Genotyping PCRs with flanking primers we designed based on the sequence near the junction between transgene and genome sequences confirmed that the presence or absence of the transgene only in the line (hAce2-Tg#16). The authors have already reported examples of transgene mapping by Genomic Walking (Exp Anim 53: 103-111, 2004) and whole genome sequencing of long reads (Exp Anim 69: 279-286, 2020). This study confirmed that short-read whole-genome sequencing was also useful for mapping transgenes and designing PCR for genotyping of transgenic mice.

Support or Funding Information

Institutional funding.