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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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Cre-mediated excision in vivo. (A): (a)–(c) Oviductstransfected with pCE-29 alone. EGFP-derived green fluorescence (but notHcRed1-derived red fluorescence) was observed. (d)–(f) Oviductstransfected with pCRTEIL alone. HcRed1-derived red fluorescence (but notEGFP-derived green fluorescence) was observed. (g)–(i) Oviductscotransfected with pCRTEIL and pCAG/NCre. Both types of fluorescence (indicatedby arrows in (h) and (i)) were seen, although green fluorescence appeared to beweaker than red fluorescence. (j) and (k) Cryostat sections of oviducts sampled1 day after introduction of pCRTEIL and pCAG/NCre DNA (0.2 μg each) and subsequent in vivo electroporation. Note thatstrong, but patchy, EGFP fluorescence presents in some oviductal epithelial cells (indicatedby arrows in (k)). (a), (d), (g), and (j) Photographs taken under light(Light); (b), (e), (h), and (k): photographs taken under UV illumination usingfilters for detection of green fluorescence (EGFP); (c), (f), and (i)photographs taken under UV illumination using filters for detection of redfluorescence (HcRed1). (B) Luciferase reporter gene activity in oviduct sampled 1day after in vivo gene deliveryto oviducts. Oviducts were in vivo transfected with pCRTEIL alone (negative control),pCRTEIL and pCAG/NCre (experiment), pCAG/NCre alone (negative control), or pCL(positive control). Oviducts were isolated from each mouse at 2 days followingelectroporation to measure luc activity. From a total of 8 females injectedwith DNA, 4 oviducts were subjected to measurement of luc activity for eachtransfection group. Note also that the vertical axis of the figure isinterrupted and has one scale. Different letters indicate statisticallysignificant differences (P < .001).
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fig3: Cre-mediated excision in vivo. (A): (a)–(c) Oviductstransfected with pCE-29 alone. EGFP-derived green fluorescence (but notHcRed1-derived red fluorescence) was observed. (d)–(f) Oviductstransfected with pCRTEIL alone. HcRed1-derived red fluorescence (but notEGFP-derived green fluorescence) was observed. (g)–(i) Oviductscotransfected with pCRTEIL and pCAG/NCre. Both types of fluorescence (indicatedby arrows in (h) and (i)) were seen, although green fluorescence appeared to beweaker than red fluorescence. (j) and (k) Cryostat sections of oviducts sampled1 day after introduction of pCRTEIL and pCAG/NCre DNA (0.2 μg each) and subsequent in vivo electroporation. Note thatstrong, but patchy, EGFP fluorescence presents in some oviductal epithelial cells (indicatedby arrows in (k)). (a), (d), (g), and (j) Photographs taken under light(Light); (b), (e), (h), and (k): photographs taken under UV illumination usingfilters for detection of green fluorescence (EGFP); (c), (f), and (i)photographs taken under UV illumination using filters for detection of redfluorescence (HcRed1). (B) Luciferase reporter gene activity in oviduct sampled 1day after in vivo gene deliveryto oviducts. Oviducts were in vivo transfected with pCRTEIL alone (negative control),pCRTEIL and pCAG/NCre (experiment), pCAG/NCre alone (negative control), or pCL(positive control). Oviducts were isolated from each mouse at 2 days followingelectroporation to measure luc activity. From a total of 8 females injectedwith DNA, 4 oviducts were subjected to measurement of luc activity for eachtransfection group. Note also that the vertical axis of the figure isinterrupted and has one scale. Different letters indicate statisticallysignificant differences (P < .001).

Mentions: Wenext examined whether this Cre-loxP system using pCRTEIL as a reporter plasmid can also be used in vivo. For this purpose, genedelivery into oviductal epithelium was performed by DNA injection into thelumen of oviducts and subsequent EP [9]. Instillation of pCE-29 plasmid yieldedbright green fluorescence throughout the ampulla ((b) in Figure 3(A))but not red fluorescence ((c) in Figure 3(A)). When pCRTEIL plasmid DNA wassingly introduced, no fluorescence for EGFP was observed ((e) in Figure 3(A)). Instead,red fluorescence was observed in some oviductal epithelial cells ((f) in Figure3(A)). Coinjection with pCRTEIL and pCAG/NCre plasmids resulted ingeneration of both red and green fluorescence, although green fluorescenceappeared to be weaker than red fluorescence (indicated by arrows in (h) and (i)in Figure 3(A)).


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)

Cre-mediated excision in vivo. (A): (a)–(c) Oviductstransfected with pCE-29 alone. EGFP-derived green fluorescence (but notHcRed1-derived red fluorescence) was observed. (d)–(f) Oviductstransfected with pCRTEIL alone. HcRed1-derived red fluorescence (but notEGFP-derived green fluorescence) was observed. (g)–(i) Oviductscotransfected with pCRTEIL and pCAG/NCre. Both types of fluorescence (indicatedby arrows in (h) and (i)) were seen, although green fluorescence appeared to beweaker than red fluorescence. (j) and (k) Cryostat sections of oviducts sampled1 day after introduction of pCRTEIL and pCAG/NCre DNA (0.2 μg each) and subsequent in vivo electroporation. Note thatstrong, but patchy, EGFP fluorescence presents in some oviductal epithelial cells (indicatedby arrows in (k)). (a), (d), (g), and (j) Photographs taken under light(Light); (b), (e), (h), and (k): photographs taken under UV illumination usingfilters for detection of green fluorescence (EGFP); (c), (f), and (i)photographs taken under UV illumination using filters for detection of redfluorescence (HcRed1). (B) Luciferase reporter gene activity in oviduct sampled 1day after in vivo gene deliveryto oviducts. Oviducts were in vivo transfected with pCRTEIL alone (negative control),pCRTEIL and pCAG/NCre (experiment), pCAG/NCre alone (negative control), or pCL(positive control). Oviducts were isolated from each mouse at 2 days followingelectroporation to measure luc activity. From a total of 8 females injectedwith DNA, 4 oviducts were subjected to measurement of luc activity for eachtransfection group. Note also that the vertical axis of the figure isinterrupted and has one scale. Different letters indicate statisticallysignificant differences (P < .001).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3: Cre-mediated excision in vivo. (A): (a)–(c) Oviductstransfected with pCE-29 alone. EGFP-derived green fluorescence (but notHcRed1-derived red fluorescence) was observed. (d)–(f) Oviductstransfected with pCRTEIL alone. HcRed1-derived red fluorescence (but notEGFP-derived green fluorescence) was observed. (g)–(i) Oviductscotransfected with pCRTEIL and pCAG/NCre. Both types of fluorescence (indicatedby arrows in (h) and (i)) were seen, although green fluorescence appeared to beweaker than red fluorescence. (j) and (k) Cryostat sections of oviducts sampled1 day after introduction of pCRTEIL and pCAG/NCre DNA (0.2 μg each) and subsequent in vivo electroporation. Note thatstrong, but patchy, EGFP fluorescence presents in some oviductal epithelial cells (indicatedby arrows in (k)). (a), (d), (g), and (j) Photographs taken under light(Light); (b), (e), (h), and (k): photographs taken under UV illumination usingfilters for detection of green fluorescence (EGFP); (c), (f), and (i)photographs taken under UV illumination using filters for detection of redfluorescence (HcRed1). (B) Luciferase reporter gene activity in oviduct sampled 1day after in vivo gene deliveryto oviducts. Oviducts were in vivo transfected with pCRTEIL alone (negative control),pCRTEIL and pCAG/NCre (experiment), pCAG/NCre alone (negative control), or pCL(positive control). Oviducts were isolated from each mouse at 2 days followingelectroporation to measure luc activity. From a total of 8 females injectedwith DNA, 4 oviducts were subjected to measurement of luc activity for eachtransfection group. Note also that the vertical axis of the figure isinterrupted and has one scale. Different letters indicate statisticallysignificant differences (P < .001).
Mentions: Wenext examined whether this Cre-loxP system using pCRTEIL as a reporter plasmid can also be used in vivo. For this purpose, genedelivery into oviductal epithelium was performed by DNA injection into thelumen of oviducts and subsequent EP [9]. Instillation of pCE-29 plasmid yieldedbright green fluorescence throughout the ampulla ((b) in Figure 3(A))but not red fluorescence ((c) in Figure 3(A)). When pCRTEIL plasmid DNA wassingly introduced, no fluorescence for EGFP was observed ((e) in Figure 3(A)). Instead,red fluorescence was observed in some oviductal epithelial cells ((f) in Figure3(A)). Coinjection with pCRTEIL and pCAG/NCre plasmids resulted ingeneration of both red and green fluorescence, although green fluorescenceappeared to be weaker than red fluorescence (indicated by arrows in (h) and (i)in Figure 3(A)).

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