NINDS 24-003326

This is required for a collaborative project between the Inherited Movement Disorders Unit (PI: Narendra) and the Computational Structural Biology Section (PI: Forrest), which has been funded as one of the special Collaborative Translational Initiatives by NINDS. This library will allow for the high throughput functional screening of mutations in the gene product Parkin, which is the leading cause of recessive Parkinson’s disease.

Mutations in Parkin are the leading cause of recessive Parkinson’s disease. However, the functional signficiance of most mutations that are present in the population is unknown. Additionally, activation of Parkin through small molecular agnoists may allow for modulation of mitophagy to protect against Parkinson’s disease and other disorders, but a comprehensive structural understanding of Parkin activation is lacking, making it difficult to rationally design drugs aimed at modulating Parkin activity. The purpose of the study is provide a comprehensive structure-function analysis of Parkin by through a combination of function assessment and structural modeling of all possible Parkin missense mutations. For the functional assessment in this project, the Parkin cDNA library will be a critical element, allowing the high throughput screening of Parkin function, using assays that have already been developed by the Parkin lab.

Independently and not as an agent of the Government, the Contractor shall furnish all the necessary services, qualified personnel, material, equipment, and facilities, not otherwise provided by the Government as needed to perform the Statement of Work below:
Custom Single Site Variant Library, Parkin_variant, 465
variant positions, all positions pooled and then cloned
into customer-supplied vector, delivered as purified
plasmd DNA

Custom Single Site Variant Library, Parkin_variant, 465 variant positions, all positions pooled and then cloned into customer-supplied vector, delivered as purified plasmid DNA. All 19 amino acid variants and a silent mutation with defined codons should be introduced at each of the 465 variant positions.
Parkin should have a N-terminal TagBFP2 fluroscent protein (FP) in frame with Parkin. Parkin should be cloned into the NheI and BamHI sites fo the pCIG3 IRES EGFP vector. An additional NheI site should be added between the TagBFP2 FP and Parkin cDNA sequence to allow removal for the FP by restriction digestion cloning.
Representation of mutations within the cloned pool must be verified by next-generation sequencing. At least 90% of the positions must pass, meaning that at least 75% of the intended mutations at that position are incorporated as seen in our NGS data.

Government will supply the sequence of the desired deliverable and will provide at least 10 ug of purified pCIG3 IRES GFP plasmid for cloning.

Contractor will provide the following deliverable within 8 weeks of receipt of the plasmid to be provided by the government and awarding of the contract (whichever is later). The deliverable is:
Custom Single Site Variant Library, Parkin_variant, 465
variant positions, all positions pooled and then cloned
into customer-supplied vector, delivered as purified
plasmid DNA

Contractor will provide will provide a electronic report at the conclusion of the project summarizing the results of the next generation sequencing data of the Custom Single Site Variant Library, including data on the frequence of each variant in the library.

The government retains the rights to publish data regarding the representation of variants within the delivered library and exclusive rights to data generating using the library. The contractor will be acknowledged as the source of the material in any publication. The government retains the right to further distribute the pooled library generated on contract with third parties.