Cell Lines & Plasmids

2T1 cell line – Alsford et al (2005) Mol Biochem Parasitol 144: 142-8
I developed this bloodstream form T. bucei cell line while working in David Horn’s lab during 2004/05; it has become the workhorse cell line for much of the tetracycline-inducible expression and RNAi work done in our labs. It is now routinely used by a number of groups in the UK, and is also used by groups in mainland Europe, South America and the USA.

The cell line expresses TetR from the pHD1313 cassette integrated at the tubulin locus – see Alibu et al (2004) Mol Biochem Parasitol 139: 75-82.

A ribosomal spacer on chromosome 2a, known to be capable of supporting high level RNA polymerase I transcription, was engineered to contain a ‘landing pad‘ (Figure, below). This recombinant locus consists of a 3’-HYG fragment and VSG expression site promoter-driven PAC ORF. Transfection with a linearised pRPa plasmid (see below) will reconsititute the HYG ORF and delete the PAC ORF, rendering the transformants resistant to hygromycin and sensitive to puromycin.

Prior to transfection, 2T1 (VSG221 expressing, Tagged, clone 1) T. b. brucei should be maintained in 1 µg/ml phleomycin (TetR) and and 1 µg/ml puromycin (‘landing pad‘).

pRP plasmid series | sequences | maps | details
A series of constructs designed to integrate at a random ribosomal spacer. Transformants will exhibit a range of insert expression levels depending on the ribosomal spacer at which they integrate. Versions are available for MYC and GFP tagging (BLA or HYG selectable markers), and stem-loop RNAi (HYG). These can be used in any T. brucei cell line that isn’t already resistant to hygromycin or blasticidin.

ph3Ep – ‘landing pad’ plasmid | sequence
This plasmid consists of a 3′-HYG fragment  and a VSG ES promoter-driven PAC ORF; it is designed to replace an integrated pRP(HYG) expression plasmid, generating a landing pad locus that can be reproducibly targeted by the pRPa plasmids (see below), and cells that are hygromycin sensitive and puromycin resistant.

For more details, see Alsford et al (2005) Mol Biochem Parasitol 144: 142-8.

pRPa plasmid series | sequences | maps | details
A series of constructs designed to integrate at a ribosomal spacer containing an integrated landing pad; the pRPa plasmids can only be used in the 2T1 cell line. These plasmids only differ from the pRP series in that they carry a 5′-HYG fragment and have AscI sites flanking the integration sequences (for linearisation prior to integration at the landing pad locus). Versions are available for MYC and GFP tagging, and stem-loop RNAi. We also have a limited number of constitutive constructs that lack the Tet operator.

pRPa transformants exhibit consistent insert expression levels, as integration always occurs at the landing pad locus (all derived lines are hygromycin resistant). However, not all lines become puromycin sensitive, suggesting that cross-over has occurred upstream of the landing pad VSG ES promoter. If you require your derived cell lines to be puromycin sensitive to enable downstream manipulations, you will need to screen your transformants – normally >50% of transformants are puromycin sensitive, though it can be less for those transformed with a stem-loop RNAi pRPa construct.

pNAT construct series
N-terminal tagging | maps | details
C-terminal tagging | maps | details
A series of GFP and multi-MYC constructs for tagging endogenous loci at the N- or C-termini. Variants carrying BLA and HYG selectable markers are available. These can be used in any T. brucei cell line that isn’t already resistant to blasticidin or hygromycin.

For more details, see Alsford & Horn (2008) Mol Biochem Parasitol 161: 76-9.

Details of additional T7 polymerase-driven tagging and RNAi plasmids are available on the Horn Lab Resources page.

If you would like to use the 2T1 cell line or any of these plasmids in your work, please feel free to contact myself or David Horn.

Some recent publications by other groups showing the utility of these tools:
Wilkinson et al (2008) Proc Natl Acad Sci USA. 105:5022-27
Bot et al (2010) Antimicrob Agents Chemother. 54:4246-52
Frearson et al (2010) Nature 464:728-32
Vincent et al (2010) PLoS Pathog. 6:e1001204
Hall et al (2011) J Biol Chem. 286: 13088-95
Obado et al (2011) Nucleic Acids Res. 39:1023-33
Hall & Wilkinson (2012) Antimicrob Agents Chemother. 56:115-23
Hall et al (2012) Antimicrob Agents Chemother. 56:5821-30
May et al (2012) Mol Microbiol. 84:77-92
Tiengwe et al (2012) PLoS One 7:e32674
Monnerat et al (2013) PLoS One 8:e67327
Taylor et al (2013) Mol Microbiol89:420-32
Trenaman et al (2013) Nucleic Acids Res. 41:943-60
Jones et al (2014 ) PLoS Pathogens 10:e1003886
Mony et al (2014) Nature 505:681-5

If you’ve used the 2T1 cell line or these plasmids, and have any comments or questions about them, please feel free to email me. Also, if you’ve modified any of the plasmids with your own preferred fluorescent or epitope tags, or cloning system,  please let me know; this will help me to maintain a comprehensive list of available plasmids, enabling others in the field to benefit from them.