Hedgehog Signaling Protocols (Methods in Molecular Biology by Jamila I. Horabin

By Jamila I. Horabin

Meant for Molecular Biologists, Biochemists, and Geneticists attracted to manipulating and studying the Hedgehog Signaling Pathway, this quantity bargains a variety of designated chapters on experimental description, together with anecdotal tips and notes.

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8% agarose-coated dishes. 5. Harvest explants when control embryos (of the same batch) have reached the desired stage. 2. Overexpression of Ptc)Loop2 by RNA Microinjection Ptc)Loop2 is a truncated version of the Hh receptor Patched1 that lacks the second extracellular loop to which the Hh protein normally binds. It was first described as a Hh-insensitive form of Patched whose expression blocks Shhinduced signal transduction in the neural tube (18). Overexpression of Ptc)Loop2 in whole embryos is performed by injection of 2 ng synthetic capped mRNA into one blastomere of two-cell stage embryos, in combination with 100 pg G-galactosidase mRNA as linage tracer.

Overexpression of Ptc)Loop2 by RNA Microinjection Ptc)Loop2 is a truncated version of the Hh receptor Patched1 that lacks the second extracellular loop to which the Hh protein normally binds. It was first described as a Hh-insensitive form of Patched whose expression blocks Shhinduced signal transduction in the neural tube (18). Overexpression of Ptc)Loop2 in whole embryos is performed by injection of 2 ng synthetic capped mRNA into one blastomere of two-cell stage embryos, in combination with 100 pg G-galactosidase mRNA as linage tracer.

Within the sending cell, Hh modification and secretion seem to depend on the availability of cholesterol. Lipid raft formation in both cells may equally be necessary for proper secretion and reception of the signal. 11. 7% formaldehyde. 12. 8) filtered through 3 mm filter). 13. 4). 14. 0) with NaOH), 1 mg/mL Torula RNA (Sigma), 100 Rg/mL Heparin (Sigma), 1× Denhardts (100× Denhardts: 2% BSA (Fluka), 38 Hollemann et al. Fig. 2. Schematic of the microinjection set-up (see text for details). 15. 16.

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