Exon structure and targeting scheme of the Peg3 locus.

(A) Schematic representations of the genomic locus of mouse Peg3 and the targeting vector. The 9 exons of Peg3 are indicated with vertical lines. The targeting vector is made of a negative selection marker (DTA, Diphtheria Toxin A), an insertion cassette and two homology hooks. The relative positions of two homology hooks are shown along with the genomic structure of Peg3 with dotted lines. The 7.1-kb insertion cassette contains promoterless galactosidase (β-Gal) and human β-actin promoter-driven neomycin resistance gene (Neo). The bottom panel shows the relative positions of PCR primers with small arrows and the predicted sizes of genomic and RT-PCR products. (B) Genomic LD (Long Distance)-PCR. Two different sets of primers successfully amplified 6.7-kb genomic fragments from the genomic DNA of a heterozygote animal (KO), but not from that of a wild-type littermate (WT), confirming the proper targeting of the insertion cassette. (C) RT-PCR. RT-PCR was performed using the total RNA from WT and KO animals. The two primer sets (β-actin and 1a/1b) amplified similar levels of products between KO and WT, but the third primer set (1a/1c) flanking the insertion cassette amplified only from WT, confirming the proper truncation of Peg3 transcription in KO. (D) Western blot analyses using the protein extracts prepared from the neonatal brains of WT and KO. This analysis revealed the absence of PEG3 protein in the pup inheriting the KO allele paternally.