A single vector containing modified cre recombinase and LOX recombination sequences for inducible tissue-specific amplification of gene expression

Abstract

The selective alteration of the genome using Cre recombinase to target the rearrangement of genes flanked by LOX recognition sequences has required the use of two separate genetic constructs in trans, one containing cre and the other containing the gene of interest flanked by LOX sites. We have developed a strategy in which both the cre recombinase gene and LOX recombination sites may be cloned within a single vector in cis. This method uses a modified form of Cre (CREM) that contains alterations to the 5' region including the introduction of a Kozak consensus sequence and insertion of a functional intron. This system allows for the inducible, tissue-specific activation or inactivation of gene expression in a single vector and can be utilized for the 300-fold amplification of gene expression from a weak promoter. This approach can be applied to targeting strategies for generating genetically altered mice and gene therapy.

Figure 1

Gene-switch strategy using cre/LOX induced translational frameshift. (A) Gene 1 flanked by LOX sites is inserted in frame within the open reading frame of gene 2 resulting in appropriate translation of Gene 1, but not Gene 2. In the presence of Cre recombinase Gene 1 is excised and the correct reading frame of Gene 2 is restored which is now expressed. (B) EGFP/β-gal gene-switch under the transcriptional control of the SV40 early promoter. Translational start site and first 18 amino acids of E.coli gtp are upstream of the floxed EGFP followed by E.coli gtp amino acids 19–47 fused in-frame to β-gal. The insertion of a cytosine at the 3′ end of LOX I results in a disruption of the reading frame of the β-gal gene. The Cre-mediated excision of the floxed EGFP restores the correct reading frame for β-gal. (C–F) Transfection of CHO-K1 cells with pSV-EGFP/β-gal results in expression of only EGFP without cre whereas co-transfection with pCMV-cre results in loss of EGFP expression but gain of β-gal expression. (C and D) Fluorescent microscopy, GFP filter; (E and F) bright field (X 320). Assays performed 48 h post-transfection.

Figure 1

Gene-switch strategy using cre/LOX induced translational frameshift. (A) Gene 1 flanked by LOX sites is inserted in frame within the open reading frame of gene 2 resulting in appropriate translation of Gene 1, but not Gene 2. In the presence of Cre recombinase Gene 1 is excised and the correct reading frame of Gene 2 is restored which is now expressed. (B) EGFP/β-gal gene-switch under the transcriptional control of the SV40 early promoter. Translational start site and first 18 amino acids of E.coli gtp are upstream of the floxed EGFP followed by E.coli gtp amino acids 19–47 fused in-frame to β-gal. The insertion of a cytosine at the 3′ end of LOX I results in a disruption of the reading frame of the β-gal gene. The Cre-mediated excision of the floxed EGFP restores the correct reading frame for β-gal. (C–F) Transfection of CHO-K1 cells with pSV-EGFP/β-gal results in expression of only EGFP without cre whereas co-transfection with pCMV-cre results in loss of EGFP expression but gain of β-gal expression. (C and D) Fluorescent microscopy, GFP filter; (E and F) bright field (X 320). Assays performed 48 h post-transfection.

Figure 1

Gene-switch strategy using cre/LOX induced translational frameshift. (A) Gene 1 flanked by LOX sites is inserted in frame within the open reading frame of gene 2 resulting in appropriate translation of Gene 1, but not Gene 2. In the presence of Cre recombinase Gene 1 is excised and the correct reading frame of Gene 2 is restored which is now expressed. (B) EGFP/β-gal gene-switch under the transcriptional control of the SV40 early promoter. Translational start site and first 18 amino acids of E.coli gtp are upstream of the floxed EGFP followed by E.coli gtp amino acids 19–47 fused in-frame to β-gal. The insertion of a cytosine at the 3′ end of LOX I results in a disruption of the reading frame of the β-gal gene. The Cre-mediated excision of the floxed EGFP restores the correct reading frame for β-gal. (C–F) Transfection of CHO-K1 cells with pSV-EGFP/β-gal results in expression of only EGFP without cre whereas co-transfection with pCMV-cre results in loss of EGFP expression but gain of β-gal expression. (C and D) Fluorescent microscopy, GFP filter; (E and F) bright field (X 320). Assays performed 48 h post-transfection.

Figure 2

Gene-switch using pCMV-EGFP/RFP vector. (A) Generation of gene-switch reporter vector pCMV-EGFP/RFP containing the floxed EGFP gene upstream of RFP. (B–G) CHO-K1 cells were transfected with pCMV-EGFP/RFP either in the absence (B and E) or presence of pCMV-cre (C, D, F and G). (B and C) Fluorescent microscopy using GFP filter; (E and F) using RFP filter; (D) bright field; (G) using dual GFP/RFP filter (X 400). Assays performed 48 h post-transfection.

Figure 3

Amplification of gene expression using a weak, tissue-specific promoter to activate transcription from a strong promoter. (A) Luciferase reporter gene vectors. SV, SV40 early promoter; CMVe-βAc, CMV enhancer, β-actin promoter. (B) PC-3 cells were transfected with the reporter constructs pPr-luc, pCMV-STOP-luc or pCMV-STOP-luc with pPr-cre. Luciferase expression increased 15-fold using this amplification strategy. Assays performed 48 h post-transfection.

Figure 3

Amplification of gene expression using a weak, tissue-specific promoter to activate transcription from a strong promoter. (A) Luciferase reporter gene vectors. SV, SV40 early promoter; CMVe-βAc, CMV enhancer, β-actin promoter. (B) PC-3 cells were transfected with the reporter constructs pPr-luc, pCMV-STOP-luc or pCMV-STOP-luc with pPr-cre. Luciferase expression increased 15-fold using this amplification strategy. Assays performed 48 h post-transfection.

Figure 4

Modification of Cre recombinase. (A) pCMV-Cre-del is the backbone of subsequent constructs. pCMV-Cre-K contains the modified 5′ untranslated region and amino acid changes as indicated. pCMV-CREM contains the chimeric/β-globin intron within the cre coding sequence. pCMV-RFP/CREM contains a floxed RFP gene within the Cre coding sequence. (B–G) Demonstration of functional activity of modified Cre. Co-transfection of pCMV-CREM with reporter plasmid pCMV-EGFP/RFP in CHO-KI cells results in the switch from EGFP to RFP expression. (B–D) Reporter pCMV-EGFP/RFP alone; (E–G) reporter with pCMV-CREM (p210). (B and E) Fluorescence with GFP filter; (C and F) fluorescence with RFP filter; (D and G) fluorescence using dual GFP/RFP filters. Assays performed 48 h post-transfection.

Figure 4

Modification of Cre recombinase. (A) pCMV-Cre-del is the backbone of subsequent constructs. pCMV-Cre-K contains the modified 5′ untranslated region and amino acid changes as indicated. pCMV-CREM contains the chimeric/β-globin intron within the cre coding sequence. pCMV-RFP/CREM contains a floxed RFP gene within the Cre coding sequence. (B–G) Demonstration of functional activity of modified Cre. Co-transfection of pCMV-CREM with reporter plasmid pCMV-EGFP/RFP in CHO-KI cells results in the switch from EGFP to RFP expression. (B–D) Reporter pCMV-EGFP/RFP alone; (E–G) reporter with pCMV-CREM (p210). (B and E) Fluorescence with GFP filter; (C and F) fluorescence with RFP filter; (D and G) fluorescence using dual GFP/RFP filters. Assays performed 48 h post-transfection.

Figure 5

Generation of probasin-cre vectors and enhanced amplification of gene expression using a second Cre. (A) Probasin driven Cre recombinase expression vectors. (B) PC-3 cells were transfected with the murine androgen receptor producing-plasmid pCMV-mAR (0.06 pmol) in combination with the indicated plasmids (0.1 pmol) in the presence or absence of 4 nM mibolerone. The presence of the second conditional cre vector led to the additional 8-fold amplification of luciferase activity. Assays performed 48 h post-transfection. (C) Time course of gene amplification using a second conditionally expressed Cre recombinase. PC-3 cell were transfected with pCMV-mAR (0.06 pmol) and the indicated plasmids. Except as indicated, 0.1 pmol of each plasmid was used in transfections. pTK-RL (0.1 pmol) (Promega) was used to normalize for transfection efficiency.

Figure 5

Generation of probasin-cre vectors and enhanced amplification of gene expression using a second Cre. (A) Probasin driven Cre recombinase expression vectors. (B) PC-3 cells were transfected with the murine androgen receptor producing-plasmid pCMV-mAR (0.06 pmol) in combination with the indicated plasmids (0.1 pmol) in the presence or absence of 4 nM mibolerone. The presence of the second conditional cre vector led to the additional 8-fold amplification of luciferase activity. Assays performed 48 h post-transfection. (C) Time course of gene amplification using a second conditionally expressed Cre recombinase. PC-3 cell were transfected with pCMV-mAR (0.06 pmol) and the indicated plasmids. Except as indicated, 0.1 pmol of each plasmid was used in transfections. pTK-RL (0.1 pmol) (Promega) was used to normalize for transfection efficiency.

Figure 5

Generation of probasin-cre vectors and enhanced amplification of gene expression using a second Cre. (A) Probasin driven Cre recombinase expression vectors. (B) PC-3 cells were transfected with the murine androgen receptor producing-plasmid pCMV-mAR (0.06 pmol) in combination with the indicated plasmids (0.1 pmol) in the presence or absence of 4 nM mibolerone. The presence of the second conditional cre vector led to the additional 8-fold amplification of luciferase activity. Assays performed 48 h post-transfection. (C) Time course of gene amplification using a second conditionally expressed Cre recombinase. PC-3 cell were transfected with pCMV-mAR (0.06 pmol) and the indicated plasmids. Except as indicated, 0.1 pmol of each plasmid was used in transfections. pTK-RL (0.1 pmol) (Promega) was used to normalize for transfection efficiency.

Figure 6

Construction and functional testing of single vectors containing both modified Cre recombinase and LOX-dependent conditional reporter cassettes. (A) pPr-CREM/CMV-STOP-luc vector is a fusion of pPr-CREM and CMV-STOP-luc cassettes. pPr-CREM/Ins/CMV-STOP-luc contains pPr-CREM/Ins separated from CMV-STOP-luc by two chicken globin insulators. (B) Evaluation of inducible and amplified gene expression using single vector constructs. PC-3 cells were transfected with pCMV-mAR in the presence or absence of 4 nM mibolerone and additional plasmids (0.1 pmol) as indicated. Assays were performed 48 h post-transfection.

Figure 6

Construction and functional testing of single vectors containing both modified Cre recombinase and LOX-dependent conditional reporter cassettes. (A) pPr-CREM/CMV-STOP-luc vector is a fusion of pPr-CREM and CMV-STOP-luc cassettes. pPr-CREM/Ins/CMV-STOP-luc contains pPr-CREM/Ins separated from CMV-STOP-luc by two chicken globin insulators. (B) Evaluation of inducible and amplified gene expression using single vector constructs. PC-3 cells were transfected with pCMV-mAR in the presence or absence of 4 nM mibolerone and additional plasmids (0.1 pmol) as indicated. Assays were performed 48 h post-transfection.