Fig 1.
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.
Fig 2.
Growth effects of Peg3DelKO allele in neonates.
(a) Male Peg3DelKO/WT were bred with female wild-type littermates to generate Peg3WT/DelKO pups with the paternal transmission of the mutant allele. Pups were genotyped and weighed at 1 day postpartum (dpp), then compared to the average weight of the litter. The number of pups belonging to each percentile weight range was then graphed to visualize the weight distribution of Peg3DelKO pups in comparison to their wild-type littermates. (b) The same series of analyses were also repeated with the pups at the weaning age (21 dpp). Representative littermates from 1 dpp and 21 dpp are shown with corresponding genotypes indicated (c,d).
Fig 3.
Peg3 conditional knockout pups: impact on growth.
Pups with conditional Peg3 knockouts were weighed and compared to the average weight within the litter. Percentile weights within litter were then separated into 10% ranges. The numbers of pups in each percentile range are shown for MMTV-driven deletion at 1 dpp (a) and 21 dpp (b) and for Nkx2.1-driven deletion at 1 dpp (c) and 21 dpp (d).
Fig 4.
Peg3 conditional knockout dams: milk letdown.
Milk retention at 10 dpp of MMTV-driven (a) and Nkx2.1-driven (b) deletions in Peg3 are shown as a function of the weight change between every two hours in the dam subtracted by the weight change between the same time points in the pups. Therefore, the weight flux shown is a measure of how much nutrient transfer is occurring from the dam to her pups at each given time interval with positive slopes indicating weight gain in the dam that is not transferred over to the pups. Conversely, negative slopes indicate a dam giving all her mass to the pups, without gaining any weight herself. The dashed black box indicates the critical point wherein reintroduction of the dams to their pups has just occurred. (c) Oxytocin-induced milk ejection was surveyed in MMTV-driven Peg3 deleted mice alongside wild-type littermate controls. Black arrows indicate ductal branches in the mammary gland.
Fig 5.
Peg3 conditional knockout mothers: nest-building behavior.
(a) Representative conditional knockout dams and wild-type littermates highlight differences observed in the nest-building behavior. (b) A graph shows the number of dams marked as good, mediocre and bad at building nests for their litters. “Good” dams are those who tore 100% of bedding material by 14 hours. “Mediocre” dams were able to use some, but not all of the bedding material by 14 hours. “Bad” dams are those that display no interest in tearing up the bedding material to insulate their pups.