(a) Scheme for Cre-loxP-mediated recombination using pCRTEIL and pCETRIZ as reporter transgenes. Before recombination, the floxed HcRed1/CAT hybrid sequence is expressed under control of the CAG promoter in cells carrying pCRTEIL, while the EGFP and luc cDNAs are silent. Similarly, the floxed EGFP/CAT hybrid sequence is expressed in cells carrying pCETRIZ, but the HcRed1 cDNA and lacZ genes are silent. Cre-mediated recombination results in deletion of the floxed sequence, and expression of the EGFP and luc cDNAs in pCRTEIL-carrying cells or expression 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: chloramphenicol acetyltransferase gene; EGFP: enhanced green fluorescent protein cDNA; IRES: internal ribosomal entry site; Luc: firefly luciferase cDNA; NCre: nuclear location signal + Cre gene; pA: poly(A) sites.

Cre-mediated excision in vivo. (A): (a)–(c) Oviducts transfected with pCE-29 alone. EGFP-derived green fluorescence (but not HcRed1-derived red fluorescence) was observed. (d)–(f) Oviducts transfected with pCRTEIL alone. HcRed1-derived red fluorescence (but not EGFP-derived green fluorescence) was observed. (g)–(i) Oviducts cotransfected with pCRTEIL and pCAG/NCre. Both types of fluorescence (indicated by arrows in (h) and (i)) were seen, although green fluorescence appeared to be weaker than red fluorescence. (j) and (k) Cryostat sections of oviducts sampled 1 day after introduction of pCRTEIL and pCAG/NCre DNA (0.2 μg each) and subsequent in vivo electroporation. Note that strong, but patchy, EGFP fluorescence presents in some oviductal epithelial cells (indicated by arrows in (k)). (a), (d), (g), and (j) Photographs taken under light (Light); (b), (e), (h), and (k): photographs taken under UV illumination using filters for detection of green fluorescence (EGFP); (c), (f), and (i) photographs taken under UV illumination using filters for detection of red fluorescence (HcRed1). (B) Luciferase reporter gene activity in oviduct sampled 1 day after in vivo gene delivery to 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 following electroporation to measure luc activity. From a total of 8 females injected with DNA, 4 oviducts were subjected to measurement of luc activity for each transfection group. Note also that the vertical axis of the figure is interrupted and has one scale. Different letters indicate statistically significant differences (P < .001).

Cre-mediated excision in vitro. (a)–(c) NIH3T3 cells transfected with pCRTEIL alone. When cells were inspected for fluorescence 1 day after transfection, HcRed1-derived red fluorescence (but not EGFP-derived green fluorescence) was predominant (indicated by arrows in (b)). (a)–(c) Microphotographs indicate the same cells. (d)–(f) NIH3T3 cells cotransfected with pCRTEIL and pCAG/NCre. Some cells predominantly exhibited green fluorescence (indicated by arrows in (e) and (f)), while other cells exhibited both types of fluorescence (indicated by arrowheads in (e) and (f)). (d)–(f) Microphotographs indicate the same cells. (g)–(j) NIH3T3 cells transfected with pCETRIZ alone. When cells were inspected for fluorescence 1 day after transfection, EGFP-derived green fluorescence (but not HcRed1-derived red fluorescence and lacZ expression) was predominant (indicated by arrows in (h)). (g)–(j) Microphotographs indicate the same cells. (k)–(n) NIH3T3 cells cotransfected with pCETRIZ and pCAG/NCre. Cells exhibited both green and red fluorescence as well as lacZ expression (indicated by arrows in (l), (m), and (n)). (k)–(n) Microphotographs indicate the same cells. (a), (d), (g), and (k) Photographs taken under light (Light); (b), (e), (i), and (m) photographs taken under UV illumination using filters for detection of red fluorescence (HcRed1); (c), (f), (h), and (l) photographs taken under UV illumination using filters for detection of green fluorescence (EGFP); (j) and (n) photographs taken under light after staining with X-Gal (lacZ). Bar: 10 μm.

Gene gun-mediated skin transfection with pCETRIZ (a)–(d) and pCETRIZ + pCAG/NCre (e)–(h). After shaving of the abdominal area of mice, plasmid DNA coated with gold particles was transfected with a 300-psi helium gas pulse using the Helios gene gun. Two days after gene delivery, skin was dissected and inspected for fluorescence under a UV illuminator. After observation for fluorescence, the same sample was then subjected to histochemical staining for lacZ activity. Figures (a)–(c) and (e)–(g) are photographs of the same portion. Figure (d) is a different portion from those in Figures (a)–(c). Similarly, Figure (h) is a different portion from those in Figures (e)–(g). (a), (d), (e), and (h) taken under light; (b) and (f): taken under UV illumination for detection of GFP fluorescence; (c) and (g): taken UV illumination for detection of red fluorescence. Bar: 10 μm.