Optical Manipulation of Xenopus Embryonic Development

Fertilized Xenopus embryos were transferred into mesh-bottomed dishes with 3% Ficoll and injected with capped, synthetic mRNAs (made using the Ambion Message Machine kit) dissolved in water at the stages indicated. The doses per cell were KRAS^G12D [71 (link)] 40pg; Arch [69 (link)], 60pg; and ChR2^D156A [72 (link)] 50pg. Two hours after injection, embryos were transferred into 0.75 × MMR for 45 minutes before they were washed and cultured in 0.1 × MMR until desired stage was reached. Injected embryos were stimulated with the appropriate wavelength of light and irradiance before or after ITLSs fully form (stages 28–35). Embryos were scored for the presence of ITLSs using bright field microscopy as described in [3 (link), 41 (link), 42 (link)].

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Chernet B.T., Adams D.S., Lobikin M, & Levin M. (2016). Use of genetically encoded, light-gated ion translocators to control tumorigenesis. Oncotarget, 7(15), 19575-19588.

Publication 2016

Message Machine kit

Embryos Ficoll Light Mesh Microscopy Mrnas Xenopus

Corresponding Organization : Tufts University

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Variable analysis

independent variables

KRAS^G12D - 40pg per cell
Arch - 60pg per cell
ChR2^D156A - 50pg per cell

dependent variables

Presence of ITLSs (intermediate target-like structures) in injected embryos

control variables

Fertilized Xenopus embryos
Embryos transferred into mesh-bottomed dishes with 3% Ficoll
Capped, synthetic mRNAs dissolved in water
Embryos transferred into 0.75 × MMR for 45 minutes after injection
Embryos cultured in 0.1 × MMR until desired stage was reached
Embryos stimulated with appropriate wavelength of light and irradiance before or after ITLSs fully form (stages 28–35)

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