Figure 1.
Tandem copies of the transgene are arrayed head-to-tail at a single insert locus.
(A) Schematic diagram of restriction enzyme sites and the sizes expected by Southern blot analysis. Bold lines show the position of the probe. B, BamHI; EI, EcoRI; S, SalI; Xb, XbaI; Xh, XhoI. (B) Southern blot analysis. Genomic DNA from MYCNTg/+ mouse was digested with indicated restriction enzymes. Note that all results are consistent with the expected size shown in (A). (C) Quantification of transgene copy number. Genomic DNA (5 µg per lane) from MYCNTg/+ and wild type mice were digested with EcoRI and SalI. Copy standards were prepared by mixing wild type DNA with a known amount of DNA and subjected to Southern blot analysis. MYCNTg/+ mouse has about 5–6 copies of the transgene.
Figure 2.
IPCR after PstI restriction enzyme digestion.
(A) Restriction enzyme map of PstI and TaqI. PstI and TaqI sites are located approximately 0.7 kb and 2.5 kb up-stream of the transgene, respectively. The bold lines indicate location of the 0.7 kb probe. Primers (OUT1 and IN1) were used for the 2nd IPCR. (B) Southern blot analysis. In addition to the expected size, an additional band (indicated by arrows) was detected by PstI (1.4 kb) and TaqI (3.5 kb) digestion. (C) PstI digested DNA fragment (1.4 kb) was eluted from the gel, purified and IPCR was carried out. The expected size (1 kb) of the 2nd PCR product was obtained.
Figure 3.
TH-MYCN transgene is integrated in a Chr18qE4 locus.
The sequence of the transgene/host DNA junctions are shown. Bold letters with underlines indicate the primers (Chr18F5 and Chr18R2) using for genotyping. PstI site is located 610 bp up-stream of the transgene. Dotted lines indicate 1,063 bp of the genomic DNA deletion. It should be noted that the deletion was shown down-stream of the transgene for convenience (see results and discussion).
Figure 4.
Genotyping PCR using flanking primer sets.
(A) The position of primers. Dotted line shows genomic deletion (see results and discussion). (B) PCR with the primers N008/N009 identifies either transgenic or non-transgenic mice, in which the product is detected as 400 bp band. On the other hand, multiple PCR with three primer sets clearly distinguish wild-type (single larger band), homozygous (single smaller band) and hemizygous (both bands) DNA. The expected size of PCR products are 500 bp and 295 bp with Chr18F1/Chr18R2/OUT1, and 1.5 kb and 1 kb with Chr18F5/R2/R3.