Photoactivation of the CreER(T2) Recombinase for Conditional Site-Specific Recombination with High Spatiotemporal Resolution

Sinha, D.K., Neveu, P., Gagey, N., Aujard, I., Le Saux, T., Rampon, C., Gauron, C., Kawakami, K., Leucht, C., Bally-Cuif, L., Volovitch, M., Bensimon, D., Jullien, L., and Vriz, S.
Full text @ Zebrafish

Cre-mediated recombination in the green-to-red reporter line. In Tg (ef1a: loxP-GFP-loxP-dsRed2) embryos injected with 3 pg of Cre mRNA at one-cell stage (A), dsRed fluorescence is ubiquitously observed at 36 hpf. Embryos were injected with 3 pg of CreERT2 mRNA, further incubated with embryo medium (B) or 1 μM cyclofen-OH (Ind) (C), and observed at 2 dpf. Variation of the recombination efficiency with the amount of injected CreERT2 mRNA (in pg) and inducer concentration: 0 μM (blue triangle) or 3 μM (red square). The percentage of embryos exhibiting dsRed fluorescence was scored by epifluorescence microscopy at 2 dpf (D). The error bars represent statistical errors estimated as √p(1-p)/n. Scale bars: 100 μm. dpf, days postfertilization.

Photocontrol of CreERT2 recombinase activity in Tg reporter line. Transgenic (ef1α:loxP-GFP-loxP-dsRed2) embryos were injected with 3 pg of CreERT2 mRNA at the one-cell stage and further incubated with 3 μM of caged inducer (cInd). Photoactivation of cInd at the 3/6-somite stage resulted in dsRed expression observed here at 2 dpf. In epifluorescence microscopy, the non-UV illuminated embryos (A) do not express dsRed, whereas they do express dsRed in every cell upon global UV illumination (B), in few cells upon twophoton excitation in a single cell in the forming retina (D), or in a just formed somite (E). Scale bars: 100 μm. Kinetics of recombination was determined by quantitative PCR (C). Genomic DNA was prepared from 30 tg (ef1α:loxP-GFP-loxPdsRed2) embryos; Green: NI, noninjected embryos; blue: embryos injected at one-cell stage with 30 pg cre-ERT2 mRNA, incubated with 3 μM of caged inducer (cInd), illuminated at 50% epiboly, and then fixed for DNA purification; red: embryos injected at one-cell stage with 30 pg cre mRNA and allowed to develop until 24 hpf. The average values (±standard error of the mean) from at least three independent experiments are presented; comparisons with t=0 were performed by a Student’s t-test. Values of p < 0.05 were considered to indicate statistical significance (***p < 0.001; **p < 0.01).

This image is the copyrighted work of the attributed author or publisher, and ZFIN has permission only to display this image to its users. Additional permissions should be obtained from the applicable author or publisher of the image. Full text @ Zebrafish