Molecular characterization of Peg3FlpKO and Peg3DelKO mouse lines.

(a) Schematic representation of Peg3 alleles. Arrows above each allele indicate transcriptional direction and length. Exons are indicated by boxes, with Exon 6 denoted as a white “6”. Flippase recognition target (FRT) sites are shown as green triangles. LoxP sites are indicated by red triangles. In the Peg3CoKO allele, we inserted a cassette containing a splice acceptor (SA) sequence, an internal ribosomal entry site (IRES) and a β-Galactosidase (β-Gal) reporter gene, followed by a poly-adenylation signal (pA). Neomycin Resistance gene (NeoR) is followed by another pA. Crossing Peg3CoKO mice with a Flp-expressing line results in the Peg3FlpKO allele. Successive crossing of Peg3FlpKO mice with Cre-expressing lines results in the Peg3DelKO allele. (b) RT-PCR of Peg3 from various tissues in Peg3FlpKO line. β-Actin was used as an internal control. (c) RT-PCR of Peg3 from various tissues in Peg3DelKO. Cre-mediated recombination of Exon 6 results in the smaller amplicon size (346 bp in length) as compared to the wild-type product (453 bp in length). (d) Western blots from the 1-day-old heads of Peg3DelKO. To visualize expression of PEG3 protein, western blots were probed for Peg3, stripped and then probed for β-Actin. Locations of primers used in this study are indicated as blue letters under each allele. The Primer legend at the bottom shows which primer corresponds to each abbreviation.