Skip to main content
. 2003 Oct;23(19):6936–6943. doi: 10.1128/MCB.23.19.6936-6943.2003

FIG. 1.

FIG. 1.

Gene targeting of the Plk2 locus. (A) Creation of a Plk2 deletion allele. Plk2 exons are indicated as solid boxes, with the asterisk indicating the first exon. The XbaI (Xb) fragment was cloned into a gene replacement vector, and the SalI fragment was replaced by a Neo cassette. The positions of PCR primers (p1 to p3) and exons whose corresponding cDNA sequences (5′ and 3′ probes) were used as the probes in Southern and Northern blot analyses are indicated. B, BamHI; S, SalI. (B) Southern blot analysis showing the disruption of the Plk2 locus. Genomic DNA was digested with BamHI and hybridized with radiolabeled 3′ Plk2 probe, followed by autoradiography. The wild-type locus yielded a fragment of ∼13 kbp, whereas the disrupted locus gave rise to a 6.8-kbp fragment, owing to a BamHI site introduced by the Neo cassette. +/+, +/−, and −/−, Plk2+/+, Plk2+/−, and Plk2−/−, respectively. (C) PCR analysis of the disrupted Plk2 locus. PCRs were carried out with primers p1 to p3 and mouse tail DNA. Reaction mixtures were resolved on an agarose gel and stained with ethidium bromide. (D) Northern blot analysis showing the disruption of Plk2 expression in Plk2−/− mice. On top is an autoradiograph showing detection with the 3′ Plk2 probe of the full-length and a truncated (arrowhead) Plk2 message in total RNA isolated from mouse brain. The controls were total RNA isolated from serum-starved NIH 3T3 cells (0h) or from cells stimulated with serum for 1 h (1h). The middle blot shows that the truncated Plk2 message does not hybridize to the 5′ Plk2 probe. The lower blot shows a methylene blue-stained membrane to demonstrate loading among the lanes. (E) Western blot analysis indicates the lack of Plk2 production in cultured embryonic fibroblasts. Cells were treated with nocodazole (M) and released into the cell cycle for 190 min (G1). On top is a Western blot with a polyclonal antibody specific for the C terminus of Plk2. The arrow indicates the position of Plk2, and molecular mass markers (in kilodaltons) are shown on the left. Below is a Western blot with anti-Erk1 antibody to demonstrate loading in each lane.