Given the right resources, nearly everything in biology is possible.

Plasmids containing coding sequences for dark ion channel genes (longest splice isoform whenever possible) were obtained in various formats from the hORFeome, and other commercial sources. For plasmids from hORFeome collection, ORFs were already in pLX304 vector, which were used directly for lentivirus production.

For plasmids in non-pLX304 backbone ion channel ORFs were cloned into pLX304 vector using Gateway and In-Fusion strategies. Reaction mixtures for all pipelines were transformed into Stbl3 bacteria and Plasmid DNA was isolated for validation of the ion channel coding part by Sanger sequencing.

Hundreds of human genes remain ignored by the scientific community, despite that many of them are likely disease causing when mutated. Why?

These constructs allow stable lentiviral transduction of sgRNA containing plasmids and generating cell lines that can be used for CRISPRi studies.

Sequences for sgRNAs against dark ion channels were chosen from the library published in “Horlbeck et al., 2016”.The sgRNAs were ligated into a lentiviral backbone that was cut using a restriction enzyme. 

PHAROS combines data from over 100 data sources so users can access, query, filter, and download data on all human protein targets.

As part of the IDG Mission of providing data for scientific community to study the dark ion channels, we compiled disease association and mouse phenotype information by mining several public databases.

These data are available below-- and if you need additional data or would be interested in collaborating on data mining related projects, please contact us.

The disease association information is harmonized from several databases, including Online Mendelian Inheritance in Man (OMIM), ClinVar, DisGeNET and Monarch Initiative database. The mouse phenotype data is compiled from the International Mouse Phenotyping Consortium (IMPC) and Mouse Genome Informatics (MGI).

An important clue to dissecting the role for a gene in health and disease is knowing the cells and tissues in which it is expressed

We characterized and quantified the expression levels of the dark genes in subsets of sensory (dorsal root ganglion; DRG) and spinal cord neurons, both in naive conditions (wild-type mice) as well as in the setting of neuropathic and inflammatory chronic pain.

We performed fluorescent multiplex in situ hybridization on DRG and spinal cord tissue harvested from wild-type mice as well as 7 days after peripheral nerve cut (neuropathic pain condition) or 3 days after hindpaw injection of complete freund’s adjuvant (inflammatory condition). Quantification was performed using Image J.