Light-controlled phosphorylation in the TrkA-Y785 site by photosensitive UAAs activates the MAPK/ERK signaling pathway

Background:This paper aims to establish a light-controlled phosphorylation detection method at the Y785 site of tropomyosin receptor kinase A(TrkA)receptor in mammalian cells by using genetic code expansion technology and detecting the effects of optical activation of this site on the downstream MAPK/ERK pathway.The study is based on the current situation that the regulatory mechanism of TrkA phosphorylation has not been fully elucidated.Methods:Two photosensitive unnatural amino acids,p-azido-L-phenylalanine(AzF)and photo-caged tyrosine(ONB)were introduced into the TrkA-Y785 site by genetic code expansion technology and site-directed mutagenesis.Western blotting and laser confocal imaging were conducted to analyze the effects of this site on activating the MAPK/ERK pathway and nerve cell differentiation before and after photostimulation.Results:Our results supplemented the light-controlled results of the TrkA-Y785 site based on our previous research and verified that Y785 also makes important contributions in regulating the MAPK/ERK pathway.Conclusion:This study demonstrated the significant contributions of the TrkAY785 site in regulating the ERK pathway by precisely controlling the phosphorylation state of a single tyrosine site.

Generation of obese rat model by transcription activator-like effector nucleases targeting the leptin receptor gene

The laboratory rat is a valuable mammalian model organism for basic research and drug discovery. Here we demonstrate an efficient methodology by applying transcription activator-like effector nucleases(TALENs) technology to generate Leptin receptor(Lepr) knockout rats on the Sprague Dawley(SD) genetic background. Through direct injection of in vitro transcribed m RNA of TALEN pairs into SD rat zygotes, somatic mutations were induced in two of three resulting pups. One of the founders carrying bi-allelic mutation exhibited early onset of obesity and infertility. The other founder carried a chimeric mutation which was efficiently transmitted to the progenies. Through phenotyping of the resulting three lines of rats bearing distinct mutations in the Lepr locus, we found that the strains with a frame-shifted or premature stop codon mutation led to obesity and metabolic disorders. However, no obvious defect was observed in a strain with an in-frame 57 bp deletion in the extracellular domain of Lepr. This suggests the deleted amino acids do not significantly affect Lepr structure and function. This is the first report of generating the Lepr mutant obese rat model in SD strain through a reverse genetic approach. This suggests that TALEN is an efficient and powerful gene editing technology for the generation of disease models.