Mammary development during pregnancy (controlateral control vs. cleared fat pad) to define transdifferentiation factors. Mus musculus

Tissue from cleared mammary fat pad and sham-operated controlateral control was removed before and at 10, 15,17, and 19 days of pregnancy. Cleared vs. controlateral tissue was hybridized in dye-swap design to in-house cDNA microarry platform. This design aims to identify factors signaling from the epithelial part of the mammary gland to the surrounding fat pad to initiate adipo-epithelial transdifferentiation. Overall design: We surgically removed the epithelial part of the 4th mammary gland (including the nipple and the rudimentary ductal tree) in three weeks old CD1 mice where the ductal anlage is confined to the proximal part of the mammary fat pad close to the nipple. The controlateral gland was sham-operated as control. This leaves a cleared mammary fat pad in its endogenous environment. The dissected tissues were subjected to whole mount analysis to judge if the removal of epithelial tissue was complete. At the age of 10 to 15 weeks the tissues were harvested before and 10, 15, 17, and 19 days after the start of pregnancy. RNA from cleared fat pads were compared to sham-operated contralateral controls by competitive hybridization on two-channel microarrays. If enough material was available three different pools of RNA (= three biological replicates) were used for hybridization (from d19 material only two replicates were obtained) in a dye-swap configuraiton (one biological replicate of d17 and d19 samples did not provide sufficient amount for a dyw-swap pair).