Optimizing Electroporation Conditions for Transfection of Zebrafish Cell Lines

1. p-Galactosidase-encoding plasmid (e.g., pcDNA3.1myc-his lacZ, Invitrogen).

2. Carrier DNA plasmid pBluescript II SK(-) (Stratagene).

Fig. 1. In vivo bioluminescence assay of a zebrafish luciferase reporter cell line. Regulation of gene expression by the circadian clock in response to light has been visualized by stably transfecting a luciferase reporter construct, driven by the zfperiod4 clock gene promoter (7). The cells are seeded into a 96-well plate and then a single addition of 0.5 mM beetle luciferin is made at the start of the assay. The assay continues uninterrupted for 20 d. The plate is counted automatically using a Packard TopCount scintillation counter. Between counts, the plates are exposed to various lighting conditions. Bioluminescence is plotted on the y-axis (counts per second) and time on the x-axis (hours from start of the experiment), where a white/black bar shows the duration of light-dark periods respectively. Apart from the extended periods of constant dark and light, each light and dark period lasts 12 h. The graph represents the mean of data obtained from 16 independent wells. The data is entirely consistent with the mRNA expression profile of the endogenous zfperiod4 gene (7).

Fig. 1. In vivo bioluminescence assay of a zebrafish luciferase reporter cell line. Regulation of gene expression by the circadian clock in response to light has been visualized by stably transfecting a luciferase reporter construct, driven by the zfperiod4 clock gene promoter (7). The cells are seeded into a 96-well plate and then a single addition of 0.5 mM beetle luciferin is made at the start of the assay. The assay continues uninterrupted for 20 d. The plate is counted automatically using a Packard TopCount scintillation counter. Between counts, the plates are exposed to various lighting conditions. Bioluminescence is plotted on the y-axis (counts per second) and time on the x-axis (hours from start of the experiment), where a white/black bar shows the duration of light-dark periods respectively. Apart from the extended periods of constant dark and light, each light and dark period lasts 12 h. The graph represents the mean of data obtained from 16 independent wells. The data is entirely consistent with the mRNA expression profile of the endogenous zfperiod4 gene (7).

3. TNE: 10 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, pH 8.0.

5. Protein assay dye reagent (Bio-Rad).

6. PM2, pH 7.0: 60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgCl2.

7. ONPG solution, pH 7.0: 8 mg/mL 0-nitrophenyl-ß-D-galactopyranoside (ONPG), 60 mM Na2HPO4, 40 mM NaH2PO4. Store at -20°C in the dark as small aliquots.

8. ß-Mercaptoethanol.

10. Gene pulser apparatus (Bio-Rad).

11. Hemocytometer.

12. 4-mm Sterile electroporation cuvets (Peqlab).

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