|
Figure 1. Dilution of electroporated plasmid reduces expression level. (A) Bulk electroporation of the Xenopus optic tectum involves the injection of a plasmid solution into the tectal ventricle followed by passing current pulses across plate electrodes that span the targeted brain region. Transduction occurs on the side toward the positive electrode due to the negative charge of plasmid DNA. (B–E) Reducing the concentration of EGFP plasmid mainly lowers the levels of expression per cell. (C–E) Two-photon z-series projections through the optic tectum of an intact Xenopus tadpole collected using the same laser excitation intensity. (C) At 2 μg/μL a large number of bright cells, especially radial glial progenitors, are labeled. (D) 0.5 μg/μL or (E) 0.1 μg/μL labels fewer cells, which are also much fainter. Scale bar, 50 μm.
|
|
Figure 2. Schematic of CREMSCLE method. (A) Electroporation of large amounts of pCALNL-EGFP containing a floxed neogenin “stop cassette†does not lead to EGFP expression unless pCAG-Cre plasmid is co-expressed. The Cre recombinase removes the stop cassette flanked by loxP sites to allow the translation of EGFP. (B) When pCAG-Cre and pCALNL-EGFP are coexpressed in roughly equimolar ratios most cells will express EGFP. As the concentration of pCAG-Cre plasmid is reduced, keeping pCALNL-EGFP levels constant, only the very few cells that express Cre recombinase will activate pCALNL-EGFP to allow high levels of EGFP expression.
|
|
Figure 3. Titration of Cre permits single cell labeling without decreasing signal intensity. Two-photon z-projections of the transfected lobe of the tadpole optic tectum demonstrate that electroporation of 1 μg/μL pCALNL-EGFP plasmid together with increasingly dilute concentrations of pCAG-Cre plasmid results in a decreasing number of labeled tectal cells with little apparent decrease in the brightness of EGFP expression. Animals were imaged (A–E) 2 and (A′–E′) 5 days after electroporation with 1 μg/μL pCALNL-EGFP plus pCAG-Cre concentrations of (A,A′) 100 ng/μL, (B,B′) 10 ng/μL, (C,C′) 1 ng/μL, (D,D′) 0.2 ng/μL, or (E,E′) 0.1 ng/μL. (F,G) Cells continue to mature and develop complex dendritic arbors over this time as can be seen in higher magnification z-series projections. Only the lowest dilution of Cre plasmid produced cells with completely non-overlapping dendritic arbors by day 5, which would be suitable for single-cell reconstruction. Scale bar, (A–E) 50 μm, (F,G) 20 μm.
|
|
Figure 4. Quantification of labeled cells imaged over three consecutive days. Tadpoles were bilaterally electroporated with 1 μg/μL pCALNL-EGFP mixed with 1.0, 0.2, and 0.1 ng/μL pCAG-Cre plasmid (n = 19, 20, 20) and subsequently screened daily for EGFP-expressing cells from 2 to 4 days post-electroporation. (A) The number of EGFP-positive cells per animal was substantially lower with decreased levels of Cre plasmid. (B) A single isolated cell could be found in nearly half of the animals from the 1.0 and 0.2 ng/μL groups on all days of imaging, but much less frequently in the 0.1 ng/μL group. In this case the 0.2 ng/μL group had the most useful optimization, with sparse expression in a large proportion of animals (high-efficiency of single cell labeling).
|
|
Figure 5. CREMSCLE tends to label more immature tectal neurons than SCE. Tadpoles were electroporated by SCE with pEGFP-N1 (1 μg/μL) or by CREMSCLE with pCAG-Cre (0.25 ng/μl) and pCALNL-EGFP (1 μg/μL), and subsequently imaged daily for EGFP-expressing cells from 2 to 5 days post-electroporation. (A) Two-photon z-projections of single cells following labeling by SCE or CREMSCLE. (B,C) Quantification of total dendritic arbor length (B) and number of dendritic branch tips (C) over 4 days of consecutive 2-photon imaging. Scale bar, 20 μm. ****p < 0.0001 for main effect, RM ANOVA, n = 7 cells per group.
|
|
Figure 6. CREMSCLE can be used for co-expression of two proteins. (A,B) Two-photon z-projections of double labeled cells at 3 days post-electroporation. (A) pCAG-Cre (0.5 ng/μL) co-electroporated with pLNL-EGFP (1 μg/μL) and (Aâ²) pCMV-mCherry (3 μg/μL) leads to (Aâ²â²) double labeling of sparse EGFP-expressing cells within a field of mCherry-labeled cells. Most cells labeled by EGFP are also positive for mCherry. (B) Co-electroporation of pCAG-Cre (0.1 ng/μL) with pLNL-EGFP (1 μg/μL) and (Bâ²) pLNL-dsRed (3 μg/μL) results in nearly all cells expressing (Bâ²â²) both EGFP and dsRed by 3 days post-electroporation. (C) Number of cells per animal that expressed EGFP fluorescence (green), dsRed fluorescence (red), or both (yellow). By 2 days post-electroporation nearly every cell was double-labeled. n = 12 animals. Scale bar, 20 μm.
|
|
FIGURE 1. Dilution of electroporated plasmid reduces expression level. (A) Bulk electroporation of the Xenopus optic tectum involves the injection of a plasmid solution into the tectal ventricle followed by passing current pulses across plate electrodes that span the targeted brain region. Transduction occurs on the side toward the positive electrode due to the negative charge of plasmid DNA. (B–E) Reducing the concentration of EGFP plasmid mainly lowers the levels of expression per cell. (C–E) Two-photon z-series projections through the optic tectum of an intact Xenopus tadpole collected using the same laser excitation intensity. (C) At 2 μg/μL a large number of bright cells, especially radial glial progenitors, are labeled. (D) 0.5 μg/μL or (E) 0.1 μg/μL labels fewer cells, which are also much fainter. Scale bar, 50 μm.
|
|
FIGURE 2. Schematic of CREMSCLE method. (A) Electroporation of large amounts of pCALNL-EGFP containing a floxed neogenin “stop cassette†does not lead to EGFP expression unless pCAG-Cre plasmid is co-expressed. The Cre recombinase removes the stop cassette flanked by loxP sites to allow the translation of EGFP. (B) When pCAG-Cre and pCALNL-EGFP are coexpressed in roughly equimolar ratios most cells will express EGFP. As the concentration of pCAG-Cre plasmid is reduced, keeping pCALNL-EGFP levels constant, only the very few cells that express Cre recombinase will activate pCALNL-EGFP to allow high levels of EGFP expression.
|
|
FIGURE 3. Titration of Cre permits single cell labeling without decreasing signal intensity. Two-photon z-projections of the transfected lobe of the tadpole optic tectum demonstrate that electroporation of 1 μg/μL pCALNL-EGFP plasmid together with increasingly dilute concentrations of pCAG-Cre plasmid results in a decreasing number of labeled tectal cells with little apparent decrease in the brightness of EGFP expression. Animals were imaged (A–E) 2 and (A′–E′) 5 days after electroporation with 1 μg/μL pCALNL-EGFP plus pCAG-Cre concentrations of (A,A′) 100 ng/μL, (B,B′) 10 ng/μL, (C,C′) 1 ng/μL, (D,D′) 0.2 ng/μL, or (E,E′) 0.1 ng/μL. (F,G) Cells continue to mature and develop complex dendritic arbors over this time as can be seen in higher magnification z-series projections. Only the lowest dilution of Cre plasmid produced cells with completely non-overlapping dendritic arbors by day 5, which would be suitable for single-cell reconstruction. Scale bar, (A–E) 50 μm, (F,G) 20 μm.
|
|
FIGURE 4. Quantification of labeled cells imaged over three consecutive days. Tadpoles were bilaterally electroporated with 1 μg/μL pCALNL-EGFP mixed with 1.0, 0.2, and 0.1 ng/μL pCAG-Cre plasmid (n = 19, 20, 20) and subsequently screened daily for EGFP-expressing cells from 2 to 4 days post-electroporation. (A) The number of EGFP-positive cells per animal was substantially lower with decreased levels of Cre plasmid. (B) A single isolated cell could be found in nearly half of the animals from the 1.0 and 0.2 ng/μL groups on all days of imaging, but much less frequently in the 0.1 ng/μL group. In this case the 0.2 ng/μL group had the most useful optimization, with sparse expression in a large proportion of animals (high-efficiency of single cell labeling).
|
|
FIGURE 5. CREMSCLE tends to label more immature tectal neurons than SCE. Tadpoles were electroporated by SCE with pEGFP-N1 (1 μg/μL) or by CREMSCLE with pCAG-Cre (0.25 ng/μl) and pCALNL-EGFP (1 μg/μL), and subsequently imaged daily for EGFP-expressing cells from 2 to 5 days post-electroporation. (A) Two-photon z-projections of single cells following labeling by SCE or CREMSCLE. (B,C) Quantification of total dendritic arbor length (B) and number of dendritic branch tips (C) over 4 days of consecutive 2-photon imaging. Scale bar, 20 μm. ****p < 0.0001 for main effect, RM ANOVA, n = 7 cells per group.
|
|
FIGURE 6. CREMSCLE can be used for co-expression of two proteins. (A,B) Two-photon z-projections of double labeled cells at 3 days post-electroporation. (A) pCAG-Cre (0.5 ng/μL) co-electroporated with pLNL-EGFP (1 μg/μL) and (Aâ²) pCMV-mCherry (3 μg/μL) leads to (Aâ²â²) double labeling of sparse EGFP-expressing cells within a field of mCherry-labeled cells. Most cells labeled by EGFP are also positive for mCherry. (B) Co-electroporation of pCAG-Cre (0.1 ng/μL) with pLNL-EGFP (1 μg/μL) and (Bâ²) pLNL-dsRed (3 μg/μL) results in nearly all cells expressing (Bâ²â²) both EGFP and dsRed by 3 days post-electroporation. (C) Number of cells per animal that expressed EGFP fluorescence (green), dsRed fluorescence (red), or both (yellow). By 2 days post-electroporation nearly every cell was double-labeled. n = 12 animals. Scale bar, 20 μm.
|
