# Neuro293: A REST-knockout HEK-293 cell line enables the expression of neuron-restricted genes for the high-throughput testing of human neurobiology and the biochemistry of neuronal proteins

## TL;DR

Researchers created a new cell line called Neuro293 by knocking out REST in HEK-293 cells, enabling the expression of neuron-related proteins for easier and faster study of neurobiology.

## Contribution

The novel REST-knockout HEK-293 cell line, Neuro293, enables rapid expression of neuron-restricted genes for high-throughput neurobiology studies.

## Key findings

- Neuro293 cells show significant upregulation of genes related to neurobiology and membrane excitability.
- Synapsin-1, Snap-25, and Kv1.2 proteins are upregulated and localized in Neuro293 processes.
- Neuro293 cells do not exhibit excitable membranes despite ion channel upregulation.

## Abstract

Efficient interrogation of neurobiology remains bottlenecked by obtaining mature neurons. Immortalized cell lines still require lengthy differentiation periods to obtain neuron-like cells, which may not efficiently differentiate and are challenging to transfect with plasmids relative to other cell lines such as HEK-293’s. To overcome challenges with limited access to cells that express mature neuronal proteins, we knocked out the RE1-silencing transcription factor (REST) from HEK-293’s to create a novel neuron-like cell, which we name Neuro293. RNA-sequencing and bioinformatics analyses revealed a significant upregulation of genes associated with neurobiology and membrane excitability including pre-/post-synaptic proteins, voltage gated ion channels, neuron-cytoskeleton, as well as neurotransmitter synthesis, packaging, and release. Western blot validated the upregulation of Synapsin-1 (Syn1) and Snap-25 as two neuron-restricted proteins, as well as the potassium channel Kv1.2. Immunocytochemistry against Neurofilament 200 kd revealed a significant upregulation and accumulation in singular processes extending from Neuro293’s cell body. Similarly, while Syn1 increased in the cell body, Syn1 protein accumulated at the ends of processes extruding from Neuro293’s. Neuro293’s express reporter-genes through the Syn1 promoter after infection with adeno-associated viruses (AAV). However, transient transfection with AAV2 plasmids led to leaky expression through promoter-independent mechanisms. Despite an upregulation of many voltage-gated ion channels, Neuro293’s do not possess excitable membranes. Collectively, REST-knockout in HEK-293’s induces a quickly dividing and easily transfectable cell line that expresses neuron-restricted and mature neuronal proteins which can be used for high-throughput biochemical interrogation, however, without further modifications neither HEK-293’s or Neuro293’s exhibit properties of excitable membranes.