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Development of CRTEIL and CETRIZ, Cre-loxP-based systems, which allow change of expression of red to green or green to red fluorescence upon transfection with a cre-expression vector.

Ohtsuka M, Warita T, Sakurai T, Watanabe S, Inoko H, Sato M - J. Biomed. Biotechnol. (2009)

Bottom Line: Single transfection with pCRTEIL resulted in predominant expression of red fluorescence.Similarly, cotransfection with pCETRIZ and pCAG/NCre resulted in change of green to red fluorescence.These noninvasive systems will be useful in cell lineage analysis, since descendants of cells exhibiting newly activated gene expression can be continuously monitored in noninvasive fashion.

View Article: PubMed Central - PubMed

Affiliation: Division of Basic Molecular Science and Molecular Medicine, School of Medicine, Tokai University, Bohseidai, Isehara, Kanagawa 259-1193, Japan. masasato@ms.kagoshima-u.ac.jp

ABSTRACT
We developed Cre-loxP-based systems, termed CRTEIL and CETRIZ, which allow gene switching in a noninvasive manner. Single transfection with pCRTEIL resulted in predominant expression of red fluorescence. Cotransfection with pCRTEIL and Cre-expression plasmid (pCAG/NCre) caused switching from red to green fluorescence. Similarly, cotransfection with pCETRIZ and pCAG/NCre resulted in change of green to red fluorescence. These noninvasive systems will be useful in cell lineage analysis, since descendants of cells exhibiting newly activated gene expression can be continuously monitored in noninvasive fashion.

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(a) Scheme for Cre-loxP-mediated recombination using pCRTEIL and pCETRIZ as reportertransgenes. Before recombination, the floxed HcRed1/CAT hybrid sequence isexpressed under control of the CAG promoter in cells carrying pCRTEIL, whilethe EGFP and luc cDNAs are silent. Similarly, the floxed EGFP/CAT hybridsequence is expressed in cells carrying pCETRIZ, but the HcRed1 cDNA and lacZgenes are silent. Cre-mediated recombination results in deletion of the floxedsequence, and expression of the EGFP and luc cDNAs in pCRTEIL-carrying cells orexpression of HcRed1 and lacZ in cells carrying pCETRIZ. (b) Plasmids (pCE-29, pCL,and pCAG/NCre) are used for expression of EGFP, luc, and NCre, respectively. All plasmids have a pBluescript SK(-) backbone. Abbreviations are CAG:cytomegalovirus enhancer + chicken β-actin promoter; CAP site: transcription start site; CAT: chloramphenicolacetyltransferase gene; EGFP: enhanced green fluorescent protein cDNA; IRES:internal ribosomal entry site; Luc: firefly luciferase cDNA; NCre: nuclearlocation signal + Cre gene; pA: poly(A) sites.
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fig1: (a) Scheme for Cre-loxP-mediated recombination using pCRTEIL and pCETRIZ as reportertransgenes. Before recombination, the floxed HcRed1/CAT hybrid sequence isexpressed under control of the CAG promoter in cells carrying pCRTEIL, whilethe EGFP and luc cDNAs are silent. Similarly, the floxed EGFP/CAT hybridsequence is expressed in cells carrying pCETRIZ, but the HcRed1 cDNA and lacZgenes are silent. Cre-mediated recombination results in deletion of the floxedsequence, and expression of the EGFP and luc cDNAs in pCRTEIL-carrying cells orexpression of HcRed1 and lacZ in cells carrying pCETRIZ. (b) Plasmids (pCE-29, pCL,and pCAG/NCre) are used for expression of EGFP, luc, and NCre, respectively. All plasmids have a pBluescript SK(-) backbone. Abbreviations are CAG:cytomegalovirus enhancer + chicken β-actin promoter; CAP site: transcription start site; CAT: chloramphenicolacetyltransferase gene; EGFP: enhanced green fluorescent protein cDNA; IRES:internal ribosomal entry site; Luc: firefly luciferase cDNA; NCre: nuclearlocation signal + Cre gene; pA: poly(A) sites.

Mentions: Thereporter plasmid pCRTEIL (Figure 1(a)) was constructed through several cloningsteps. First, pCRT-17, an intermediate product for pCRTEIL, was constructed byinserting an 8.8-kb fragment containing HcRed1 cDNA + poly(A) sites of the SV40gene isolated from pHcRed1-N1 (Clontech Laboratories, Inc., Palo Alto, Calif,USA) in front of the 5′ end of CAT in the pCAG-CAT-lacZ [4] from which the lacZgene had already been removed. A DNA fragment containing EGFP cDNA, IRES, andluc cDNA was then placed immediately downstream of the loxP site located at the 3′ end of the CAT gene in pCRT-17 toobtain pCRTEIL-6. The resulting pCRTEIL has a backbone of pBluescript SK(-)(Stratagene, La Jola, Calif, USA). The reporter plasmid pCETRIZ (Figure 1(a))was constructed in a multistep process. First, pACS, an intermediate productcarrying “I-Sce I-Sfi I-Xba I-Spe I-Sty I-Nhe I-Sfi I-I-Sce I” cassette, was generated byintroducing linker oligo into a pBluescript II-based vector. A fragmentcontaining IRES, lacZ gene, and poly(A) sites was introduced into a Sty I site (which had been blunt) ofpACS to generate pACSIZA. Next, pCETIZ was generated by ligation of a 4.5-kb Spe I fragment containing CAG promoter, floxed EGFP cDNA and CATgene, and poly(A) sites into an Xba Isite of pACSIZA. Finally, pCETRIZ was constructed by introducing PCR-amplifiedHcRed1 cDNA into a Spe I site of pCETIZ.


Development of CRTEIL and CETRIZ, Cre-loxP-based systems, which allow change of expression of red to green or green to red fluorescence upon transfection with a cre-expression vector.

Ohtsuka M, Warita T, Sakurai T, Watanabe S, Inoko H, Sato M - J. Biomed. Biotechnol. (2009)

(a) Scheme for Cre-loxP-mediated recombination using pCRTEIL and pCETRIZ as reportertransgenes. Before recombination, the floxed HcRed1/CAT hybrid sequence isexpressed under control of the CAG promoter in cells carrying pCRTEIL, whilethe EGFP and luc cDNAs are silent. Similarly, the floxed EGFP/CAT hybridsequence is expressed in cells carrying pCETRIZ, but the HcRed1 cDNA and lacZgenes are silent. Cre-mediated recombination results in deletion of the floxedsequence, and expression of the EGFP and luc cDNAs in pCRTEIL-carrying cells orexpression of HcRed1 and lacZ in cells carrying pCETRIZ. (b) Plasmids (pCE-29, pCL,and pCAG/NCre) are used for expression of EGFP, luc, and NCre, respectively. All plasmids have a pBluescript SK(-) backbone. Abbreviations are CAG:cytomegalovirus enhancer + chicken β-actin promoter; CAP site: transcription start site; CAT: chloramphenicolacetyltransferase gene; EGFP: enhanced green fluorescent protein cDNA; IRES:internal ribosomal entry site; Luc: firefly luciferase cDNA; NCre: nuclearlocation signal + Cre gene; pA: poly(A) sites.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2664460&req=5

fig1: (a) Scheme for Cre-loxP-mediated recombination using pCRTEIL and pCETRIZ as reportertransgenes. Before recombination, the floxed HcRed1/CAT hybrid sequence isexpressed under control of the CAG promoter in cells carrying pCRTEIL, whilethe EGFP and luc cDNAs are silent. Similarly, the floxed EGFP/CAT hybridsequence is expressed in cells carrying pCETRIZ, but the HcRed1 cDNA and lacZgenes are silent. Cre-mediated recombination results in deletion of the floxedsequence, and expression of the EGFP and luc cDNAs in pCRTEIL-carrying cells orexpression of HcRed1 and lacZ in cells carrying pCETRIZ. (b) Plasmids (pCE-29, pCL,and pCAG/NCre) are used for expression of EGFP, luc, and NCre, respectively. All plasmids have a pBluescript SK(-) backbone. Abbreviations are CAG:cytomegalovirus enhancer + chicken β-actin promoter; CAP site: transcription start site; CAT: chloramphenicolacetyltransferase gene; EGFP: enhanced green fluorescent protein cDNA; IRES:internal ribosomal entry site; Luc: firefly luciferase cDNA; NCre: nuclearlocation signal + Cre gene; pA: poly(A) sites.
Mentions: Thereporter plasmid pCRTEIL (Figure 1(a)) was constructed through several cloningsteps. First, pCRT-17, an intermediate product for pCRTEIL, was constructed byinserting an 8.8-kb fragment containing HcRed1 cDNA + poly(A) sites of the SV40gene isolated from pHcRed1-N1 (Clontech Laboratories, Inc., Palo Alto, Calif,USA) in front of the 5′ end of CAT in the pCAG-CAT-lacZ [4] from which the lacZgene had already been removed. A DNA fragment containing EGFP cDNA, IRES, andluc cDNA was then placed immediately downstream of the loxP site located at the 3′ end of the CAT gene in pCRT-17 toobtain pCRTEIL-6. The resulting pCRTEIL has a backbone of pBluescript SK(-)(Stratagene, La Jola, Calif, USA). The reporter plasmid pCETRIZ (Figure 1(a))was constructed in a multistep process. First, pACS, an intermediate productcarrying “I-Sce I-Sfi I-Xba I-Spe I-Sty I-Nhe I-Sfi I-I-Sce I” cassette, was generated byintroducing linker oligo into a pBluescript II-based vector. A fragmentcontaining IRES, lacZ gene, and poly(A) sites was introduced into a Sty I site (which had been blunt) ofpACS to generate pACSIZA. Next, pCETIZ was generated by ligation of a 4.5-kb Spe I fragment containing CAG promoter, floxed EGFP cDNA and CATgene, and poly(A) sites into an Xba Isite of pACSIZA. Finally, pCETRIZ was constructed by introducing PCR-amplifiedHcRed1 cDNA into a Spe I site of pCETIZ.

Bottom Line: Single transfection with pCRTEIL resulted in predominant expression of red fluorescence.Similarly, cotransfection with pCETRIZ and pCAG/NCre resulted in change of green to red fluorescence.These noninvasive systems will be useful in cell lineage analysis, since descendants of cells exhibiting newly activated gene expression can be continuously monitored in noninvasive fashion.

View Article: PubMed Central - PubMed

Affiliation: Division of Basic Molecular Science and Molecular Medicine, School of Medicine, Tokai University, Bohseidai, Isehara, Kanagawa 259-1193, Japan. masasato@ms.kagoshima-u.ac.jp

ABSTRACT
We developed Cre-loxP-based systems, termed CRTEIL and CETRIZ, which allow gene switching in a noninvasive manner. Single transfection with pCRTEIL resulted in predominant expression of red fluorescence. Cotransfection with pCRTEIL and Cre-expression plasmid (pCAG/NCre) caused switching from red to green fluorescence. Similarly, cotransfection with pCETRIZ and pCAG/NCre resulted in change of green to red fluorescence. These noninvasive systems will be useful in cell lineage analysis, since descendants of cells exhibiting newly activated gene expression can be continuously monitored in noninvasive fashion.

Show MeSH