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Figure 5 | BMC Molecular Biology

Figure 5

From: Cloning-free regulated monitoring of reporter and gene expression

Figure 5

Cloning-free conversion of genes into inducible-repressible expression cassette. (A) Schematic representation of the cloning-free conversion of a gene product into tetracycline-inducible gene. Forward primer contains 3' end sequences that targets a minimal region in the vector template and 5'end that contain two or three copies of TetO sites (Table 1, SEQ. 10–12). Reporter-C' MODC denotes unstable EGFP version that was created by fusing EGFP with MODC C-terminus as described in Methods. (B) HeLa Tet-off cells in 96-well plates were transfected with 75 ng of PCR products generated from reporter vector (A) and subsequently treated with or without 0.1 ug/ml of doxycycline for 26 hr. As a control, EGFP PCR product which lacks TetO was used. Arrows denotes % reduction. (C) Purified PCR products amplified from expression vectors that contain either wild type EGFP or unstable EGFP were transfected onto HeLa Tet-off cells in the presence or absence of 0.1 ug of doxycycline for 26 hrs. Data in B and C are Mean ± SEM (quadruplicate) from a representative experiment of three. (D) Representative images of the effect of doxycycline on the TetO-containing reporter expression PCR products with or without normalization of RFP-expressing PCR product that lack TetO sites. Images represent single (I) or co-transfection (II) experiments. (E) HeLa Tet-off cells were transfected with purified PCR products that were amplified from TTP expression vector with TetO2 forward primer (Table 1, SEQ. 11) and polyA reverse primer, in the absence or presence of 0.1 ug/ml of doxycycline. Western blotting using anti-body to TTP and GAPDH was performed. V denotes vector only.

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