Ywhae gene = 14-3-3 epsilon = tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, epsilon polypeptide (Ywhae)
Mus musculus tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, epsilon polypeptide (Ywhae), mRNA NCBI Reference Sequence: NM_009536.4
Dr. Yi Zhou's 14-3-3 FKO (Thy1-YFP-DIfopein) Transgenic Mice
See SFN abstracts (2010) 53.1/M17 and (2011) 790.09/FF16
From Kourtney Graham's Honor's Thesis:
In order to study the role of 14-3-3 proteins in the nervous system_ in vivo_, we have successfully created lines of neuronal-specific transgenic mice that may inhibit 14-3-3 dependent cellular processes. These transgenic mice express a YFP-fused peptide (R18) which binds to endogenous 14-3-3, and thus blocking its capability to interact with its binding partners; and are therefore considered to be a functional knockout. The transgenes are expressed under the control of the Thy-1 promoter which is neuronal-specific and produces variation of transgene expression in different brain regions, which is great among founder lines but minimal among descendants of a single founder (Feng et al. 2000). To minimize strain-dependent genetic variation, we have backcrossed the founder lines with wild-type C57BL/6 mice for at least 8 generations.
From Sun et al PMID 23861400 : Glutamatergic kainate receptors (KAR) KAR-EPSCs at mossy fiber-CA3 synapses decay significantly faster in the 14-3-3 functional knock-out mice (but no difference in NMDA-R or AMPA-R EPSC in same synapses)
R18 is a synthetic 21 amino acid unphosphorylated peptide (MW 2309.6) which binds to the same amphipathic groove on the surface of 14-3-3 as phosphorylated peptides, with high affinity for all 7 mammalian isoforms of 14-3-3 PMID 10493820. Commercial source of peptide Enzo #BML-P214-0001.
Difopein is a construct with two R18 monomers in a single 62 amino acid peptide PMID 11577088. Commercial source of peptide: Tocris #1245
Pro-His-Cys-Val-Pro-Arg-Asp-Leu-Ser-Trp-Leu-Asp-Leu-Glu-Ala- Asn-Met-Cys-Leu-Pro-OH PHCVPRDLSWLDLEANMCLP
Difopein sequence (with individual R18 monomers in parentheses):
SADGA (PHCVP RDLSW LDLEA NMCLP) GAAGL DSADG A(PHCV PRDLS WLDLE ANMCL P) GAAG LE
C54 (consisting of the carboyl-terminal 54-amino acid region of theHMG-box binding protein Ste11p ) is another 14-3-3 inhibitor that is structurally unrelated to R18/difopein. The C54 region permits sequestration of Ste11p in the cytoplasm to inhibit nuclear localization PMID 12697825.
YFP is 239 amino acids long (717 bp) according to plasmid map of Invitrogen Vivid ColorsTM pcDNATM/6.2 N-terminal EmGFP/YFP-DEST vectors see p, 28 of the manual. Difopein is 62 amino acids long (186 bp), so total YFP-difopein fusion protein should be ~ 301 amino acids (903 bp).
14-3-3 FKO papers
Vogt, Peggy, "Exploring the Function of 14-3-3 Proteins in Neurodegenerative Disease" (2011). Honors Theses. Paper 42.
C. Sun, H. Qiao, Q. Zhou, Y. Wang, Y. Wu, Y. Zhou, et al., Modulation of GluK2a Subunit-containing Kainate Receptors by 14-3-3 Proteins, J Biol Chem. 288 (2013) 24676–24690. PMID 23861400
Qiao H, Foote M, Graham K, Wu Y, Zhou Y. 14-3-3 proteins are required for hippocampal long-term potentiation and associative learning and memory. J Neurosci. 34(2014):4801-8. PMID 24695700
Antisera for IP Experiments
anti-pan-14-3-3 (K-19) , rabbit polyclonal, Santa Cruz #sc-629, 200 ug/1 ml, $279 datasheet
anti-HDAC4 (H-92), rabbit polyclonal, Santa Cruz #sc-11418, 200 ug/1 ml, $279 datasheet
anti-Torc1 (C71D11), rabbit mAb, Cell Signaling #2587 $225 datasheet