Transgenic
The present invention relates to methods and compositions for high content drug screening in Caenorhabditis elegans which may be used to identify compounds that treat disorders associated with protein aggregation. It is based, at least in part, on the discovery that Caenorhabditis elegans , genetically modified to create a model system for disorders of protein aggregation, could be used, in a high throughput screening system, to identify agents that reduce the amount of aggregated protein.
1. A Caenorhabditis elegans whose genome comprises a first transgene comprising: (a) a nucleic acid encoding a first fluorescent protein and (b) a nucleic acid encoding a human protein with a tendency to aggregate, or a polymerizable portion thereof, operably linked to a first Caenorhabditis elegans promoter, wherein said nucleic acid encoding a human protein with a tendency to aggregate, or a polymerizable portion thereof, comprises one or more synthetic introns resembling Caenorhabditis elegans introns, wherein said one or more synthetic introns comprise an AGGUAAGU splice acceptor sequence, a CAGG splice donor sequence, or both; wherein the human protein with a tendency to aggregate, or a polymerizable portion thereof, is fused with the first fluorescent protein and is expressed as a fusion protein, and wherein the expression of the fusion protein results in a detectable accumulation of the human protein in the Caenorhabditis elegans.
2. The Caenorhabditis elegans of claim 1 , whose genome further comprises a second transgene comprising a marker construct comprising a marker gene encoding a marker protein operably linked to a second Caenorhabditis elegans promoter, wherein the expression of the transgene comprising the marker construct results in a single detectable region per Caenorhabditis elegans.
3. The Caenorhabditis elegans of claim 2 , wherein the marker protein is a second fluorescent protein.
4. The Caenorhabditis elegans of claim 3 , wherein the first fluorescent protein and the second fluorescent protein are not the same.
5. The Caenorhabditis elegans of claim 2 , wherein the second Caenorhabditis elegans promoter is selected from the group consisting of a neuron-specific, a gut-specific promoter, a muscle-specific promoter, a pharynx-specific promoter and a tail-specific promoter.
6. The Caenorhabditis elegans of claim 5 , wherein the second Caenorhabditis elegans promoter is selected from the group consisting of a pharynx-specific promoter and a tail-specific promoter.
7. The Caenorhabditis elegans of claim 1 , wherein the protein is selected from the group consisting of huntingtin, synuclein, neuroserpin, ubiquitin, neurofilament protein, alpha B crystallin, and tau.
8. The Caenorhabditis elegans of claim 1 , wherein the first Caenorhabditis elegans promoter is selected from the group consisting of a neuron-specific promoter, a gut-specific promoter, a muscle-specific promoter, a pharynx-specific promoter and a tail-specific promoter.
9. The Caenorhabditis elegans of claim 1 , wherein the nucleic acid encodes an a1-antitrypsin mutant protein selected from the group consisting of ATZ, Siiyama and Mmalton, and wherein said first Caenorhabditis elegans promoter is a gut specific promoter.
10. The Caenorhabditis elegans of claim 1 , wherein the one or more synthetic intron is between 48 and 51 base pairs in length.
11. The Caenorhabditis elegans of claim 1 , wherein the protein is amyloid beta or a polymerizable portion thereof.
12. The Caenorhabditis elegans of claim 1 , wherein the protein is synuclein or a polymerizable portion thereof.
13. The Caenorhabditis elegans of claim 1 , wherein the protein is huntingtin or a polymerizable portion thereof.
14. The Caenorhabditis elegans of claim 1 , wherein the protein is tau protein or a polymerizable portion thereof.
15. The Caenorhabditis elegans of claim 1 , wherein the protein is neuroserpin or a polymerizable portion thereof.
16. The Caenorhabditis elegans of claim 1 , wherein the protein is an al-antitrypsin mutant protein selected from the group consisting of ATZ, Siiyama and Mmalton, or a polymerizable portion thereof.