MMTV Gag-GFP expression is enhanced by the insertion of the 5’UTR of MMTV or CTE of M-PMV cis-regulatory elements.

<p>(<b>A</b>) Schematic representation of the expression vectors p-EGFP, p-Gag-GFP, p-UTR-Gag-GFP and p-Gag-GFP-CTE. The approximate size of each predicted protein is shown. All vectors contained the human cytomegalovirus immediate early promoter (CMV) and a polyadenylation signal (A)n. (<b>B</b>, <b>C</b>) HEK293T cells were transfected with mock (lane 1), MMTV (lane 2), Gag-GFP (lane 3), UTR-Gag-GFP (lane 4) and Gag-GFP-CTE (lane 5) plasmids two days prior to lysis. Proteins were resolved on SDS-PAGE and detected through western blot using anti-MMTV CA (anti-CA) or anti-GFP antibodies, using anti-β-actin as a loading control. Black arrows point to the bands corresponding to expected proteins (names included beside arrows). (<b>D</b>) HEK293T cells were transfected with the respective plasmids two days prior to cytoplasmic RNA extraction. Gag-GFP mRNA was quantified with RT-qPCR. Relative expression of the Gag-GFP mRNA to β-actin mRNA (ΔCt) in the cytoplasmic fraction is shown on the <i>y-</i>axis (n = 3).