Vectors for gene expression in trypanosomes

The following tables show suitable vectors for inducible and constitutive expression in trypanosomes.

All systems involve trypanosomes expressing the Tn10 tet repressor. The promoter driving expression can be the EP1 RNA polymerase I promoter or the T7 promoter. For the plasmids with the T7 promoter, you need trypanosomes expressing T7 polymerase. In addition, in the cell lines C and H, repressor expression is driven by T7 polymerase.

For more information about the pLEW vectors from the Cross laboratory please refer to his web site (http://tryps.rockefeller.edu/), which also offers excellent advice and information and has a natty picture of swimming trypanosomes.

For vectors for expression of ds RNA please first contact Elisabetta Ullu (http://info.med.yale.edu/intmed/infdis/ullu.html)

Please note: These vectors do not work at all in other organisms, including bacteria, yeast and mammals.

The papers 1,2 3 and 5 give detailed instructions for use of these plasmids. Papers 6 and 7 illustrate their use both in in vitro cultures and in animals. They also describe some of the problems that can arise.

References:

1.	Biebinger, S., L. E. Wirtz, and C. E. Clayton. 1997. Vectors for inducible over-expression of potentially toxic gene products in bloodstream and procyclic Trypanosoma brucei. Mol. Biochem. Parasitol. 85:99-112.
2.	Wirtz, E., M. Hoek, and G. A. M. Cross. 1998. Regulated processive transcription of chromatin by T7 RNA polymerase in Trypanosoma brucei. Nucl. Acids. Res. 26:4626-4634.
3.	Wirtz, E., S. Leal, C. Ochatt, and G. A. M. Cross. 1999. A tightly regulated inducible expression system for conditional gene knock-outs and dominant-negative genetics in Trypanosoma brucei. Mol. Biochem. Parasit. 99:89-102.
4.	Wirtz, L. E., C. Hartmann, and C. E. Clayton. 1994. Gene expression mediated by bacteriophage T3 and T7 RNA polymerases in transgenic trypanosomes. Nucleic Acids Res. 22:3887-3894.
5.	Wirtz, L. E., and C. E. Clayton. 1995. Inducible gene expression in trypanosomes mediated by a procaryotic repressor. Science 268:1179-1183.
6.	van Deursen FJ, Shahi SH, C.M.R. T, Hartmann C, Guerra-Giraldez C, Matthews KR, Clayton CE. Characterisation of the growth and differentiation in vivo and in vitro of bloodstream-form Trypanosoma brucei strain TREU 927. Mol Biochem Parasitol 2001;112:163-172.
7.	Alibu P, Storm L, Haile S, Clayton C, Horn D. A doubly inducible system for RNA interference and rapid RNAi plasmid construction in Trypanosoma brucei. Mol Biochem Parasitol 2004;139:75-82

Trypanosomes

	Stage/Strain	Plasmids	Gene 1	Gene2	Marker1	Marker2		refs
A	BF 427	pHD 328	T7 pol			HYG	Normal T7 expression	4
B	BF 427	pLEW 13	T7 pol		NEO		Normal T7 expression	2,3
C	BF427	pLEW114hyg5´ pHD 328	T7 pol	TETR	NEO		Normal T7 expression Good TETR expression	2,3
D	BF 427	pHD 449		TETR	BLE		Normal TETR expression	1
E	Pro 427	pHD 328	T7 pol			HYG	Normal T7 expression	4
F	Pro 427	pHD 449		TETR	BLE		Good TETR expression	1
G	Pro 427	pLEW13 pLEW 29	T7 pol	TETR	NEO	HYG	Normal T7 expression Good TETR expression	2,3
H	BF 427	pHD 1313		2TETR		BLE	2x TETR expression	7
I	BF 927	pHD 1313		2TETR		BLE	2x TETR expression	7
J	BF 427	pHD 1313, pHD514	T7 pol	2TETR	NEO	BLE	2x TETR expression Normal T7 expression	7
K	PF 427	pHD 1313, pHD514	T7 pol	2TETR	NEO	BLE	2x TETR expression Normal T7 expression	7
L	PF 427	pHD 1313, pHD 1333	T7 pol	2TETR	NEO	BLE	2x TETR expression Inducible T7 expression	7

T7 polymerase and Tet repressor

pHD 1313	TUB	TETR	VSG3'	BLE	TETR	ACT3´	TETRexpression in both stages
pHD 328.	TUB	T7POL	ALD3^TM	HYG		ACT3	T7 polymerase expression
pHD 514	TUB	T7POL	ALD3^TM	NEO		ACT3	T7 polymerase expression
pHD 1333	spRRNA,PEPTi	T7POL	ALD3^TM	NEO		ACT3'	T7 polymerase, inducible

Inducible EP1 promoter

pLEW 100	spRRNA	ALD3^TM	<-LUC	<-P_EP^Ti	T7	BLE
pLEW 79	spRRNA	ALD3^TM	<-LUC	<-P_EP^Ti	T7	BLE
pHD 1145	spRRNA	P_EP^Ti	linker	VSG3^TM	PVSG	HYG
pHD 1146	spRRNA	P_EP^Ti	linker	ACT3^TM	PVSG	HYG
pHD 988	spRRNA	P_EP^Ti	linker	ACT3^TM	PVSG	NEO
pHD 615*	spRRNA	P_EP^Ti	CAT	VSG3^TM	PVSG	HYG
pHD 617*	spRRNA	P_EP^Ti	CAT	ACT3^TM	PVSG	HYG
pHD 597*	spRRNA	P_EP^Ti	CAT	EP3^TM	PRRNA	HYG
pHD 676*	spRRNA	P_EP^Ti	linker	ACT3^TM	PRRNA	HYG
pHD 677*	spRRNA	P_EP^Ti	linker	VSG3^TM	PVSG	HYG
pHD 678*	spRRNA	P_EP^Ti	linker	ACT3^TM	PVSG	HYG
pHD 1247*	spRRNA	P_EP^Ti	CAT	CT3^TM	PRRNA	PAC
pHD 1248*	spRRNA	P_EP^Ti	linker	VSG3^TM	PRRNA	PAC
pHD 1336	spRRNA	P_EP^Ti	linker	ACT3^TM	PVSG	BLA
pHD 1419*	177	P_EP^Ti	linker	ACT3^TM	PRRNA	HYG

*NOTES
The background and induced levels are extremely variable, depending on the repressor cell line and the gene you are expressing.
The pLEW vector combinations tend to be unstable in the absence of drug selection. We usually maintain our cells containing pHD vectors in the presence of drug also, but we have not noticed any loss of plasmids in the absence of selection. The pHD inducible system is appropriate for use in animal experiments (see reference 6 above).
Always test several clones for expression and inducibility. If you are turning off an essential gene, or over-expressing a toxic gene, you must expect a loss of regulation after a few days. No-one has yet found any way round this. See references 6 and 7 for details.
Wirtz & Cross claim that pLEW100 gives reliably lower background than 615. We haven't looked at this.
* These plasmids also contain a T7 promoter which ends up downstream of the marker and inducible cassette after integration. We have not generally found this to be a problem even in T7-pol cells. However instead of 677 and 678 you can use 1145 and 1146.

Plasmids with a VSG 3'-UTR give extremely low background and some low inducible expression in procyclics, while those with an EP1 3'-UTR have similar characteristics in bloodstream forms. If you need an inducible KO of a very poorly expressed gene, these could be your answer.

T7 expression -

Strongest expression. In inducible constructs, the background ground expression may be acceptable unless the product is extremely toxic. We find the leakage from the promoters with tet operators gives enough expression to select clones in the absence of tetracycline. Expression from pHD1667 in bloodstream forms is higher than with the actin 3'-UTR.

pLEW82	spRRNA	PT7 Ti	ALD3^TM	BLE		Expression under T7 control , 2 tet operators.
pHD 774	spRRNA	PT7Ti	CAT	EP3^TM	HYG	T7 expression in Pros. One tet operator, 5-15 x regulated
pHD 789	spRRNA	PT7Ti	CAT	ACT3^TM	HYG	T7 expression in BS or pros. One tet operator, 5-15 x regulated
pHD 1342	spRRNA	PT7Ti	CAT	EP3^TM	PAC	T7 expression in Pros. One tet operator, 5-15 x regulated
pHD 1667	spRRNA	PT7Ti	CAT	PGKC 3'	PAC	Strong expression in BS. Two tet operators, 5-10x regulated

Plasmids for constitutive expression

							Expression
pHD 330*	TUB		ALD5'	CAT	DALD3'	ACT5'	HYG	medium
pHD 1034	spRRNA	PRRNA	EP1 5'	CAT	ACT3	ACT5'	PAC	high?
pHD1462	TUB	P_T7	EP1	CAT	ACT3'	ACT5'	PAC	high with T7

TAP AND MYC TAGS

pHD 918	spRRNA	P_EP^Ti	inkerand TAP tag	ACT3^TM	PVSG	HYG
pHD 1700	spRRNA	P_EP^Ti	linkerand 2 myc tags, N-terminal	ACT3^TM		HYG
pHD 1701	spRRNA	P_EP^Ti	linkerand 2 myc tags, C-terminal	ACT3^TM		HYG

To obtain the TAP tag vector you must first sign the EMBL/Cellzome materials transfer agreement. Contact us for details.

pHD1700 and pHD1701 were made by Frank Voncken, please contact him for details and references at F.Voncken@hull.ac.uk.

Inducible RNAi with double T7

								Background	Expression	Cells
P2T7TA	spRRNA	T7term	P_T7^Ti->	link	<- PT7^Ti	T7term	HYG	Moderate	good	pol & TETR T7
P2T7TA177	177	T7term	P_T7^Ti->	link	<- P_T7^Ti	T7term	HYG	Very low		T7pol & TETR
P2T7TA177	177	T7term	P_T7^Ti->	link	<- P_T7^Ti	T7term	BLE	Very low	?	T7pol & TETR
P2T7TAblu	spRRNA	T7term	P_T7^Ti->	link	<- PT7^Ti	T7term	HYG	Moderate	good	T7pol & TETR

Plasmids for testing 3'-UTRs

	target	promoter	5'-UTR	gene	3'-UTR	5'-UTR	selection
pHD1424	spRRNA	P_T7	EP1	CAT	linker	ACT5'	PAC
pHD1437	TUB	P_T7	EP1	CAT	linker	ACT5'	PAC

References for these - see reference 7 above.