Biology subjects

Chen, B. E.

Publications and source records attributed to Chen, B. E..

3 recordsLinked to original sources

bioRxiv2024

PXGS: a Poly-Transgene Expression System based on Mutually Exclusive Splicing of Dscam

Biologists often need to investigate multiple genes simultaneously in an organism. However, it is currently not possible to express more than a few transgenes in an animal under conditional control. Here, we developed a technique based on the mutually exclusive splicing of the Down Syndrome Cell Adhesion Molecule1 (Dscam1) gene in Drosophila melanogaster to achieve simultaneous transgene expression of 12 genes at a time. We show that the hypervariable Dscam1 exon 4 region maintains its alternative splicing when placed in a UAS expression vector. Each of the twelve exon 4 alternates can be replaced with an exogenous gene of at least 10 kilobases and will express properly in vivo all under conditional genetic control. We demonstrate the expression of four different fluorophores placed in different exon 4 alternate positions in neural and non-neural cells in vivo. We validated the technique by rewiring Drosophila sensory neuron axons in vivo by simultaneously expressing several cell surface receptors within the neuron. This technology will also enable Drosophila melanogaster as a model system for synthetic biology research.

cell biology↗

bioRxiv2024

A Drug Screening Platform for Protein Expression Levels in Neurological Disorders

Neurological and psychiatric diseases and disorders affect more than half of the population. Many of these diseases are caused by the malfunctioning of protein synthesis, where too little or too much production of a protein harms a cell and its functions within the brain. We developed a drug screening platform to identify compounds that target the primary cause of these diseases, namely protein expression amounts. This cellular assay monitors protein expression of a target disease gene along with the protein expression of a control gene using the Protein Quantitation Ratioing (PQR) technique. PQR tracks protein concentration using fluorescence. We used human cells and CRISPR-Cas9 genome editing to insert the Protein Quantitation Reporter into target genes. These cells are used in high-throughput drug screening measuring the fluorescence as the assay. Drug hits can be validated using the same PQR technique or animal models of the disease. HighlightsO_LIThe assay can identify drugs that directly address the molecular cause of a disease. C_LIO_LIThe Protein Quantitation Ratioing (PQR) technique allows for tracking and measuring protein amounts over time in single living cells before, during, and after drug administration. C_LIO_LIGenome editing to insert the PQR into the target gene allows tracking of endogenous protein expression. C_LIO_LIUsing human cell lines allow for faster production of knock-in cells. C_LIO_LIPatient mutations can be replicated using genome editing during the knock-in step. C_LIO_LIUsing induced pluripotent stem cells allow for an unlimited supply of genome edited differentiated cells such as neurons with the PQR knock-in reporter. C_LI

neuroscience↗

bioRxiv2021

Tracking and Measuring Local Protein Synthesis In Vivo

Detecting when and how much a protein molecule is synthesized is important for understanding cell function, but current methods have poor cellular or temporal resolution or are destructive to cells (Dahm et al., 2008). Here, we developed a technique to detect and quantify subcellular protein synthesis events in real time in vivo. This Protein Translation Reporting (PTR) technique uses a genetic tag that produces a stoichiometric ratio of a small peptide portion of a split fluorescent protein and the protein of interest during protein synthesis. We show that the split fluorescent protein peptide can generate fluorescence within milliseconds upon binding the larger portion of the fluorescent protein, and that the fluorescence intensity is directly proportional to the number of molecules of the protein of interest synthesized. Using PTR, we tracked and measured protein synthesis events in single cells over time in vivo. We use split red fluorescent protein to detect multiple genes or alleles in single cells simultaneously. We also split a photoswitchable fluorescent protein to photoconvert the reconstituted fluorescent protein to a different channel and arbitrarily reset the time of detection of synthesis events, continually over time.

cell biology↗