Plants | Free Full-Text | A Method for Electroporation of Cre Recombinase Protein into Intact Nicotiana tabacum Cells

Figure 1.
Schematic of Cre reporter design. GFP, pA, and FLuc indicate green fluorescent protein, terminator polyadenylation signal, and firefly luciferase, respectively. Before recombination, the reporter gene is transcribed to synthesize GFP (black arrow) but not FLuc (X). Cre protein catalyzes the recombination between the two loxP sites flanking the GFP coding sequence, resulting in FLuc expression that exhibits luminescence upon addition of D-luciferin. The red arrows are the primer-binding sites for genomic PCR.

Figure 1.
Schematic of Cre reporter design. GFP, pA, and FLuc indicate green fluorescent protein, terminator polyadenylation signal, and firefly luciferase, respectively. Before recombination, the reporter gene is transcribed to synthesize GFP (black arrow) but not FLuc (X). Cre protein catalyzes the recombination between the two loxP sites flanking the GFP coding sequence, resulting in FLuc expression that exhibits luminescence upon addition of D-luciferin. The red arrows are the primer-binding sites for genomic PCR.

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Figure 2.
Electroporation-mediated Cre protein delivery into BY-2 cells. (a) Effect of poring pulse conditions on the viability of BY-2-xGxFL cells. CCK8 assay was performed 2 d after electroporation in electroporated BY-2-xGxFL cells using different poring pulse conditions: field strength (50, 100, 150, 200, or 250 V/cm), duration (10 or 20 ms) by measuring the absorbance at 450 nm. Control indicates untreated BY-2-xGxFL cells. Values shown are the mean  ±  SE of n  =  3. (b) Effect of poring pulse conditions on electroporation efficiency. Firefly luciferase activity was determined by the luminescence measurement of catalyzed D-luciferin in electroporated BY-2-xGxFL cells using different poring pulse conditions: field strength (50, 100, 150, 200, or 250 V/cm), duration (10 or 20 ms). In all cases, 5 μM Cre protein dissolved in Opti-MEMI was used for electroporation. Control indicates untreated BY-2-xGxFL cells. Values shown are the mean  ±  SE of n  =  3. (c) Effect of Cre protein concentration on on electroporation efficiency. Firefly luciferase activity as determined by the luminescence measurement of catalyzed D-luciferin in electroporated BY-2-xGxFL cells with different concentrations of Cre protein dissolved in Opti-MEMI; 0.1, 0.2, 0.5, 1.0, 2.0, or 5.0 μM. Values shown are the mean  ±  SE of n  =  3. (d) Viability of electroporated BY-2-xGxFL cells with different concentrations of Cre protein. CCK8 assay was performed 2 d after electroporation by measuring the absorbance at 450 nm. Control indicates untreated BY-2-xGxFL cells. Values shown are the mean  ±  SE of n  =  3. (e) Microchip electrophoresis analysis of genomic DNA PCR products from BY-2-xGxFL cells. The representative image in three independent experiments is shown. The 1708 bp and 552 bp fragments represent the reporter gene cassette before and after Cre-mediated recombination, respectively.

Figure 2.
Electroporation-mediated Cre protein delivery into BY-2 cells. (a) Effect of poring pulse conditions on the viability of BY-2-xGxFL cells. CCK8 assay was performed 2 d after electroporation in electroporated BY-2-xGxFL cells using different poring pulse conditions: field strength (50, 100, 150, 200, or 250 V/cm), duration (10 or 20 ms) by measuring the absorbance at 450 nm. Control indicates untreated BY-2-xGxFL cells. Values shown are the mean  ±  SE of n  =  3. (b) Effect of poring pulse conditions on electroporation efficiency. Firefly luciferase activity was determined by the luminescence measurement of catalyzed D-luciferin in electroporated BY-2-xGxFL cells using different poring pulse conditions: field strength (50, 100, 150, 200, or 250 V/cm), duration (10 or 20 ms). In all cases, 5 μM Cre protein dissolved in Opti-MEMI was used for electroporation. Control indicates untreated BY-2-xGxFL cells. Values shown are the mean  ±  SE of n  =  3. (c) Effect of Cre protein concentration on on electroporation efficiency. Firefly luciferase activity as determined by the luminescence measurement of catalyzed D-luciferin in electroporated BY-2-xGxFL cells with different concentrations of Cre protein dissolved in Opti-MEMI; 0.1, 0.2, 0.5, 1.0, 2.0, or 5.0 μM. Values shown are the mean  ±  SE of n  =  3. (d) Viability of electroporated BY-2-xGxFL cells with different concentrations of Cre protein. CCK8 assay was performed 2 d after electroporation by measuring the absorbance at 450 nm. Control indicates untreated BY-2-xGxFL cells. Values shown are the mean  ±  SE of n  =  3. (e) Microchip electrophoresis analysis of genomic DNA PCR products from BY-2-xGxFL cells. The representative image in three independent experiments is shown. The 1708 bp and 552 bp fragments represent the reporter gene cassette before and after Cre-mediated recombination, respectively.

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