doi: 10.1016/j.molbiopara.2007.03.012.
Epub 2007 Mar 27.
Functional genomics in Trypanosoma brucei: a collection of vectors for the expression of tagged proteins from endogenous and ectopic gene loci
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- PMID: 17512617
- PMCID: PMC2705915
- DOI: 10.1016/j.molbiopara.2007.03.012
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Functional genomics in Trypanosoma brucei: a collection of vectors for the expression of tagged proteins from endogenous and ectopic gene loci
Mol Biochem Parasitol.
2007 Jul.
No abstract available
Figures
Diagram showing the structure of the tetracycline-regulated transgene expression vectors described herein. The HindIII and BamHI cloning sites are indicated. Red, EP1 procyclin promoter; yellow, tet operators; lilac, T7 promoter; blue, transgene 5′ and 3′UTR; green, ribosomal RNA promoter; TY, TY epitope tag. The plasmids p2586 and p2585 were intermediates in the production of the N-terminal tagging vectors. To produce the C-terminal tagging vectors, pLEW100 was digested with BamHI and the tags inserted as BamHI BglII fragments. For the N-terminal tagging vectors, pLEW100 was digested with HindIII and a synthetic polylinker containing an XhoI site inserted to produce p2586. The resultant vector allowed directional insertion of tags as XhoI HindIII fragments. The identity and veracity of all inserts was confirmed by sequencing. pDex377 was derived from pLEW100 by replacement of the targeting sequence and the selectable marker including its promoter. DNA containing the 177 bp repeat targeting sequence was removed from p2T7-177 as a PvuI–KpnI fragment (blunted at the KpnI site with Klenow) and cloned into PvuI SacII (blunt) digested pLEW100. The resulting construct, pDex177, was digested with KpnI, blunted with Klenow, then digested with SalI and then ligated to a SalI–SphI (blunt) fragment containing the rRNA promoter and HygR gene from p2T7tiTA (kind gift of David Horn, London School of Hygiene and Tropical Medicine, UK). The sequence of pDex377 was confirmed by sequencing the whole plasmid to 4× coverage. The pDex377-derivatives were made in the same way as the pLEW100 derivatives (see above), p2585 was the intermediate plasmid for N-terminal tagging vectors. pDex577 was derived from p2T7-177 and pLEW100. The luciferase gene, encompassing 5′ and 3′UTRs and two tetracycline operators were amplified from pLEW100 with KpnI and XhoI sites incorporated into the 5′ and 3′ primers, respectively. This KpnI–XhoI fragment was then cloned into p2T7-177 digested with XhoI and KpnI. eGFP was amplified from pGad8 , the 5′ primer was designed to incorporate a HindIII site followed by a start codon and an in-frame TY epitope that was followed by a SpeI site which was immediately upstream of the second codon from eGFP. The 3′ primer also incorporated an in-frame TY epitope (and stop codon) between XbaI and BamHI sites. This PCR product was cloned as a HindIII BamHI fragment into the p2T7-177/pLEW100 hybrid vector cut with HindIII and BamHI to remove the luciferase gene. Subsequent fluorescent protein genes were cloned into the SpeI XbaI sites to replace the eGFP. For the amplification of open reading frames, the standard primer design was: forward primer, 5′-AAGCTT CCGCCACCATG followed by the next 15 bases of the ORF. The HindIII site and the initiation codon are underlined. Reverse primer: 5′-GGATCC AGAACC followed by reverse complement of last 18 bases of the ORF. The BamHI site is underlined, the spacer sequence was replaced with a stop codon if required. All amplified ORFs were cloned and sequenced prior to subcloning into expression vectors.
Diagram showing design and use of endogeneous locus tagging vectors. Green arrow, selectable marker gene; red, targeted open reading frame; yellow, 5′ sequences of targeted open reading frame. N-terminal tagging vectors were derived from pN-PTPpuro by digesting with BstBI and NotI to remove the tubulin intergenic region, an internal HindIII site, the RPA 5′UTR and PTP tag which were replaced with a polylinker containing BstBI, XhoI, HindIII, EcoRV and NotI sites. Next, the β to α tubulin inter-ORF region was cloned into the BstBI and XhoI sites. Third, a HindIII EcoRV stuffer fragment was cloned into the HindIII and EcoRV sites and finally a range of tags, as XhoI HindIII fragments were cloned into the XhoI and HindIII sites. C-terminal tagging vectors were derived from pC-PTPneo by digesting with Acc65I and EcoRI to remove the targeting sequence and tag and replacing with a polylinker containing Acc65I, SwaI, SmaI, BamHI and EcoRI sites. Next, the neomycin phosphotransferase open reading frame, which contained an internal BamHI site, was removed using NdeI and BstBI and replaced with a neomycin phosphotransferase open reading frame without a BamHI site. Third, the range of tags available as BamHI BglII fragments were cloned into the BamHI site and finally a SwaI BamHI stuffer fragment was cloned into the SwaI BamHI sites. pEnT5 is a derivative of pGad8 and pDex577. pGad8-tubulin was cut with XhoI and PvuII, blunt-ended and self ligated. This vector was then cut with XbaI and SpeI and self ligated (creating pGad8-tubulin-A). pDex577 was cut with NheI, blunt-ended and then cut with HindIII. The fragment containing the TY-tagged eGFP and 3′UTR was cloned into the pGad8-tubulin-A cut with SmaI and HindIII, creating pEnT5. The blasticidin marker gene was amplified by PCR with EcoRI and NcoI sites incorporated into the 5′ and 3′ primers, respectively. This PCR product was cloned into pEnT5 cut with NcoI and EcoRI (creating pEnT5-1). The actin 5′ intergenic region was then amplified from genomic DNA incorporating SphI and EcoRI sites on the 5′ and 3′ primers, respectively. This digested fragment was cloned into pEnT5-1 cut with SphI and EcoRI (pEnT5-2). The actin 3′ intergenic region was amplified from genomic DNA incorporating NcoI and AvrII sites on the 5′ and 3′ primers, respectively. The digested fragment was cloned into pEnT5-2 cut with NheI and NcoI to create pEnT6B. The puromycin resistance marker gene was amplified by PCR using primers incorporating EcoRI and NcoI sites in the 5′ and 3′ primers, respectively. This digested fragment was cloned into the pEnT6B cut with NcoI and EcoRI to create pEnT6P.
(A) Western blot showing GPI-PLC expression from an endogeneous locus-tagged GPI-PLC gene in a heterozygote. (B) Western blot showing the tetracycline-inducible expression of four transgenes from p2216 (Table 1) a pLEW100 derivative adding a C-terminal EYFP tag. (C) Western blot showing the tetracycline-inducible expression of a toxic transgene from pDex377, tandem HA-tagged ubiquitin. (D) Subcellular localisation of DHH1-EYFP and SCD6-CherryFP expressed from two tetracycline-inducible transgenes in the same cell line.
References
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