The pups emerge tail-first, and their cephalofoils remain folded until after birth to facilitate passage through the cloaca.
For genotyping, genomic DNA was extracted from the head (embryos) or tail (pups) and amplified by PCR according to the Jackson Lab protocol.
Around the age of 3 to 6 months old start laying drags and fooling with the pup with squirrel tails, hides or dead squirrels.
Blood cells, ear and tail of pups and each tissue of neonates killed at 15.5 or 18.5 d.p.c. were lysed in 50 mM Tris-HCl, pH 8.2, containing 25 mM EDTA, 0.5% SDS, 10 mM NaCl and 1 mg/ml proteinase K. PCR was performed with the following conditions: EGFP, 95°C 30 s, 58°C 30 s, 72°C 20 s (40 cycles) and GAPDH, 95°C 30 s, 60°C 30 s, 72°C 30 s (25 cycles).
For newborns (P0.5) and juveniles (P17), pups were sacrificed and tail DNA was used for genotyping as previously described [ 19].
Eleven pups (0.66% of all the pups born) had a cleft palate and 10 pups (0.78% of the registered pups, 0.60% of all the pups born) had a kinked tail.
According to Mendel's law of segregation, the expected outcome of a cross (mating) between a normal (homozygous) mouse and a heterozygous mutant mouse would be a litter in which one-half of the pups had spotted tails and the other half did not.
These behavioural changes were accompanied by physical alterations, which included piebald coats, floppy ears in pups, and curled tails, similar to traits that distinguish domestic dogs from wolves.
After 15 min of recording possible USIs, the pup was lifted and its tail gently pressed between thumb and index finger for releasing distress calls (DCs), which were audible to the human observer, for a minimum of 10 seconds.
Prior to behavioral assessment, pups were given head and tail markings to distinguish individuals.
