Cloning strategies are described in Table S1 . Luciferase assay was performed as described (Bayramov et al., 2011 (link)). Embryos were injected at 2–4 cell stage with synthetic c-answer + fgf8 mRNAs or solely fgf8 mRNA (3–4 nL of 100ng/μl mRNA water solution per embryo) mixed with one of the luciferase reporter plasmids: AP-1-Luc pGL4.44[luc2P/AP1 RE/Hygro] (Promega) sensitive to the stress-activated MAPK/JNK pathway, or SRE-Luc pGL4.33[luc2P/SRE/Hygro] (Promega) sensitive to MAP/ERK pathway, and the reference pRenilla plasmid (50 pg/embryo of each reporter plasmid). Animal cap explants were excised from the injected embryos at stage 10, cultured until stage 11, selected in three replicate samples by 10 explants in each and processed for luciferase analysis according to Promega protocol.
Sre luc pgl4.33 luc2p sre hygro
Manufactured by Promega
The SRE-Luc pGL4.33[luc2P/SRE/Hygro] is a reporter vector that contains the serum response element (SRE) upstream of the luciferase reporter gene luc2P. It is designed to monitor the activity of serum response factor (SRF) in eukaryotic cells.
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2 protocols using sre luc pgl4.33 luc2p sre hygro
1
Cloning and Luciferase Assay for Embryonic Signaling
2
Cloning and Luciferase Assay for Embryonic Signaling
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Cloning strategies are described in Table S1 . Luciferase assay was performed as described (Bayramov et al., 2011 (link)). Embryos were injected at 2–4 cell stage with synthetic c-answer + fgf8 mRNAs or solely fgf8 mRNA (3–4 nL of 100ng/μl mRNA water solution per embryo) mixed with one of the luciferase reporter plasmids: AP-1-Luc pGL4.44[luc2P/AP1 RE/Hygro] (Promega) sensitive to the stress-activated MAPK/JNK pathway, or SRE-Luc pGL4.33[luc2P/SRE/Hygro] (Promega) sensitive to MAP/ERK pathway, and the reference pRenilla plasmid (50 pg/embryo of each reporter plasmid). Animal cap explants were excised from the injected embryos at stage 10, cultured until stage 11, selected in three replicate samples by 10 explants in each and processed for luciferase analysis according to Promega protocol.
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