Ruprecht-Karls-Universitšt Heidelberg

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 ALD3TM HYG ACT3 T7 polymerase expression
pHD 514 TUB T7POL ALD3TM NEO ACT3 T7 polymerase expression
pHD 1333 spRRNA,PEPTi T7POL ALD3TM NEO ACT3' T7 polymerase, inducible


Inducible EP1 promoter

pLEW 100 spRRNA ALD3TM <-LUC <-PEPTi T7 BLE
pLEW 79 spRRNA ALD3TM <-LUC <-PEPTi T7 BLE
pHD 1145 spRRNA PEPTi linker VSG3TM PVSG HYG
pHD 1146 spRRNA PEPTi linker ACT3TM PVSG HYG
 pHD 988 spRRNA PEPTi linker ACT3TM PVSG NEO
 pHD 615* spRRNA PEPTi CAT VSG3TM PVSG HYG
pHD 617* spRRNA PEPTi CAT ACT3TM PVSG HYG
pHD 597* spRRNA PEPTi CAT EP3TM PRRNA HYG
pHD 676* spRRNA PEPTi linker ACT3TM PRRNA HYG
pHD 677* spRRNA PEPTi linker VSG3TM PVSG HYG
pHD 678* spRRNA PEPTi linker ACT3TM PVSG HYG
pHD 1247* spRRNA PEPTi CAT CT3TM PRRNA PAC
pHD 1248* spRRNA PEPTi linker VSG3TM PRRNA PAC
pHD 1336 spRRNA PEPTi linker ACT3TM PVSG BLA
pHD 1419* 177 PEPTi linker ACT3TM 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 ALD3TM BLE Expression under T7 control , 2 tet operators.
pHD 774 spRRNA PT7Ti CAT EP3TM HYG T7 expression in Pros. One tet operator, 5-15 x regulated
pHD 789 spRRNA PT7Ti CAT ACT3TM HYG T7 expression in BS or pros. One tet operator, 5-15 x regulated
pHD 1342 spRRNA PT7Ti CAT EP3TM 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 PT7 EP1 CAT ACT3' ACT5' PAC high with T7


TAP AND MYC TAGS

pHD 918 spRRNA PEPTi inkerand TAP tag ACT3TM PVSG HYG
pHD 1700 spRRNA PEPTi linkerand 2 myc tags, N-terminal ACT3TM HYG
pHD 1701 spRRNA PEPTi linkerand 2 myc tags, C-terminal ACT3TM 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 PT7Ti-> link <- PT7Ti T7term HYG Moderate good pol & TETR T7
P2T7TA177 177 T7term PT7Ti-> link <- PT7Ti T7term HYG Very low T7pol & TETR
P2T7TA177 177 T7term PT7Ti-> link <- PT7Ti T7term BLE Very low ? T7pol & TETR
P2T7TAblu spRRNA T7term PT7Ti-> link <- PT7Ti T7term HYG Moderate good T7pol & TETR


Plasmids for testing 3'-UTRs



target promoter 5'-UTR gene 3'-UTR 5'-UTR selection
pHD1424 spRRNA PT7 EP1 CAT linker ACT5' PAC
pHD1437 TUB PT7 EP1 CAT linker ACT5' PAC

References for these - see reference 7 above.










































© Copyright Universität Heidelberg. Impressum.