A high-throughput protein tagging toolkit that retains endogenous UTRs for studying gene regulation in Kinetoplastids.
Carbajo CG, Han X, Savur B, Upadhyaya A, Taha F, Tinti M, Wheeler RJ, Yates PA, Tiengwe C
bioRxiv [Preprint]. 2024 Nov 3:2024.11.02.621556. doi: 10.1101/2024.11.02.621556.
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PubMed
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Article
The pRExT2A plasmids serve as PCR templates for generating endogenous tagging homologous repair templates in Trypanosoma brucei. Similar to the PCR Only Tagging (pPOT) system, these plasmids contain standardized primer binding sites for PCR amplification, but uniquely incorporate a T2A peptide sequence between the tag and drug resistance marker. The plasmids come in both N- and C-terminal configurations with various combinations of tags (mNeonGreen, mScarlet, 3xTy) and drug resistance markers (BSD, PAC). The resulting PCR products can be transfected directly into cells for CRISPR/Cas9-mediated integration at endogenous loci, with the T2A peptide enabling co-expression of both the tagged protein and selection marker from a single transcript. These plasmids contain primer binding sites compatible with pPOTv6 and pPOTv7 (see Unlocking Trypanosome Biology: A Comprehensive Protein-Tagging Toolkit for Localization and Functional Analysis). Further, the pRExT2A plasmid templates contain no kinetoplastid-specific sequences, so they are fully compatible with mammalian and other eukaryotic systems.
Plasmids from Article
| ID | Plasmid | Purpose |
|---|---|---|
| 232200 | pRExT2A-NT-PmNG | Endogenous tagging of genes by CRISPR/Cas9-mediated homologous recombination; for N-terminal tagging: PAC-3xTy-T2A-3xTy-mNeonGreen |
| 232201 | pRExT2A-CT-PmNG | Endogenous tagging of genes by CRISPR/Cas9-mediated homologous recombination; for C-terminal tagging: mNeongreen-3xTy-T2A-PAC-3xTy |
| 232202 | pRExT2A-NT-PTy | Endogenous tagging of genes by CRISPR/Cas9-mediated homologous recombination; for N-terminal tagging: PAC-3xTy-T2A-3xTy |
| 232203 | pRExT2A-CT-PTy | Endogenous tagging of genes by CRISPR/Cas9-mediated homologous recombination; for C-terminal tagging: 3xTy-T2A-PAC-3xTy |
| 232206 | pRExT2A-NT-BTy | Endogenous tagging of genes by CRISPR/Cas9-mediated homologous recombination; for N-terminal tagging: BSD-3xTy-T2A-3xTy |
| 232207 | pRExT2A-CT-BmNG | Endogenous tagging of genes by CRISPR/Cas9-mediated homologous recombination; for C-terminal tagging: mNeongreen-3xTy-T2A-BSD-3xTy |
| 232208 | pRExT2A-CT-BmSc | Endogenous tagging of genes by CRISPR/Cas9-mediated homologous recombination; for C-terminal tagging: mScarlet-3xTy-T2A-BSD-3xTy |
| 232209 | pRExT2A-CT-BTy | Endogenous tagging of genes by CRISPR/Cas9-mediated homologous recombination; for C-terminal tagging: 3xTy-T2A-BSD-3xTy |
| 232548 | pRExT2A-NT-PmSc | Endogenous tagging of genes by CRISPR/Cas9-mediated homologous recombination; for N-terminal tagging: PAC-3xTy-T2A-3xTy-mScarlet |