4.3. qRT-PCR to Verify Cre-Mediated Recombination

B6.129-Pparg^tm2Rev/J mice have two loxP sites flanking exons 1 and 2 of the mouse Pparg gene [31]. Two C57.Pparg-/-^epi mice were euthanized along with a wildtype sibling control and the dorsal epidermis was removed. Adult primary keratinocyte were isolated from the tail skin of mice sacrificed at 6–15 weeks as previously described [83]. Isolated cells were cultured in supplemented EpiLife media (Thermo Fisher, Waltham, MA, USA) on pre-coated rat tail collagen type I (Thermo Fisher, Waltham, MA, USA) tissue culture plates. Confluent keratinocytes were induced to undergo terminal differentiation by increasing the CaCl₂ to 0.2mM for 72 h. RNA was then isolated using the Qiazol (Qiagen, Germantown, MD, USA) reagent and RNeasy Mini column clean-up. RNA was treated with DNase I (Thermo Fisher) and cDNA synthesized using SuperScriptIII First –Strand Synthesis System for RT-PCR (Thermo Fisher, Waltham, MA, USA). RT-PCR was then performed using primers that flank the two loxP sites: sense (5′-GTCACGTTCTGACAGGACTGTGTGAC-3′) and antisense (5′-TATCACTGGAGATCTCCGCCAACAGC-3′) [31]. These primers produce amplified products that distinguish the full-length (700-bp) and recombined (300-bp) transcripts [31].