Fig. 5. Expression of Hoxa5 in HepG2 cells and in liver of control and transgenic mice. (A) Total RNA from HepG2 cells was reverse-transcribed and PCR-amplified using two different couples of primers CR3-CR4 (378 bp) and CR7-CR8 (180 bp) specific for Hoxa5 and recognizing both human and mouse transcripts. (B) PolyA+ RNAs from liver of control (CTR) or transgenic (TG) mice (3.5 weeks), and from E9.5 transgenic embryos (positive control for expression of Hoxa5m) were reverse-transcribed and PCR-amplified using three different couples of primers. CR7-CR8 primers amplify a 180 bp fragment, demonstrating the presence of endogenous Hoxa5 mRNA in control liver and of endogenous Hoxa5 plus transgene-encoded Hoxa5m mRNAs in transgenic livers and embryos. The Hoxa5m transgene-specific CR5-CR6 primers amplify a 286 bp fragment only in transgenic livers and embryo. No amplification is obtained in absence of reverse transcriptase (RT–). First lane, molecular mass markers; last lane, PCR performed in the same conditions on H₂O instead of cDNA samples. (C) Liver polyA+ RNAs from control and transgenic mice (1-month-old) were analyzed by quantitative RT-PCR. Equivalence of signal intensity between liver Hoxa5 mRNAs (lower band) and the added standard synthetic RNA (upper band) is obtained at 40 pg for control mice (CTR, arrowhead), and 80 pg for transgenic mice (TG, arrowhead), demonstrating a twofold increase in Hoxa5 mRNA content in transgenic animals.