Biology subjects

Lee, P.-T.

Publications and source records attributed to Lee, P.-T..

3 recordsLinked to original sources

bioRxiv2018

An expanded toolkit for gene tagging based on MiMIC and scarless CRISPR tagging in Drosophila

We generated new genetic tools to efficiently tag genes in Drosophila. Double Header (DH) utilizes intronic MiMIC/CRIMIC insertions to generate artificial exons for GFP mediated protein trapping or T2A-GAL4 gene trapping in vivo based on CRE recombinase to avoid embryo injections. DH significantly increases integration efficiency compared to previous strategies and faithfully reports the expression pattern of genes and proteins. The second technique targets genes lacking coding introns using a two-step cassette exchange. First, we replace the endogenous gene with an excisable compact dominant marker using CRISPR making a null allele. Second, the insertion is replaced with a protein::tag cassette.\n\nThis sequential manipulation allows the generation of numerous tagged alleles or insertion of other DNA fragments that facilitates multiple downstream applications. Both techniques allow precise gene manipulation and facilitate detection of gene expression, protein localization and assessment of protein function, as well as numerous other applications.

genetics↗

bioRxiv2018

Loss-of-function in IRF2BPL is associated with neurological phenotypes

The Interferon Regulatory Factor 2 Binding Protein Like (IRF2BPL) gene encodes a member of the IRF2BP family of transcriptional regulators. Currently the biological function of this gene is obscure, and the gene has not been associated with a Mendelian disease. Here we describe seven individuals affected with neurological symptoms who carry damaging heterozygous variants in IRF2BPL. Five cases carrying nonsense variants in IRF2BPL resulting in a premature stop codon display severe neurodevelopmental regression, hypotonia, progressive ataxia, seizures, and a lack of coordination. Two additional individuals, both with missense variants, display global developmental delay and seizures and a relatively milder phenotype than those with nonsense alleles. The bioinformatics signature for IRF2BPL based on population genomics is consistent with a gene that is intolerant to variation. We show that the IRF2BPL ortholog in the fruit fly, called pits (protein interacting with Ttk69 and Sin3A), is broadly expressed including the nervous system. Complete loss of pits is lethal early in development, whereas partial knock-down with RNA interference in neurons leads to neurodegeneration, revealing requirement for this gene in proper neuronal function and maintenance. The nonsense variants in IRF2BPL identified in patients behave as severe loss-of-function alleles in this model organism, while ectopic expression of the missense variants leads to a range of phenotypes. Taken together, IRF2BPL and pits are required in the nervous system in humans and flies, and their loss leads to a range of neurological phenotypes in both species.

genetics↗

bioRxiv2017

Drug discovery to counteract antinociceptive tolerance with mu-opioid receptor endocytosis

Morphine antinociceptive tolerance is highly correlated with its poor ability to promote mu-opioid- receptor (MOR) endocytosis. Our objective was to discover a novel positive allosteric modulator of MOR to enhance morphine-induced MOR endocytosis. We used high-throughput screening to identify several cardiotonic steroids as positive allosteric modulators of morphine-induced MOR endocytosis having high potency and efficacy, independently of Na+/K+-ATPase inhibition. Convallatoxin was found to enhance morphine-induced MOR endocytosis through an adaptor protein 2/clathrin-dependent mechanism without regulating G protein- or {beta}-arrestin-mediated pathways. Both F243 and I292 residues of MOR were essential to the effect of convallatoxin on MOR endocytosis. Co-treatment with chronic morphine and convallatoxin reduced morphine tolerance in animal models of acute thermal pain and chronic inflammatory pain. Acute convallatoxin administration reversed morphine tolerance in morphine-tolerant mice. These findings suggest that cardiotonic steroids are potentially therapeutic for morphine side effects and open a new avenue for the study of MOR trafficking.

neuroscience↗