Regulatable retroviral vector systems


RetroTet ART(Retroviral Tetracycline-regulated Activator and Repressor Together).
RetroTet ART is a tripartite system of an activator (RetroTet RTAb(+)), a repressor (one of the RetroTet RTRg(-) vectors), and named for the "messenger of the gods", a Hermes regulatable vector such as Hermes HRSpuro-GUS. This system permits tetracycline regulated repression and activation of the gene of interest using a transrepressor that binds in the absence of tetracycline or doxycycline and a transactivator that binds in the presence of tetracycline.

The use of these vectors should be cited as follows: Rossi, F. M., Guicherit, O. M., Spicher, A., Kringstein, A. M., Fatyol, K., Blakely, B. T., & Blau, H. M. (1998). Tetracycline-regulatable factors with distinct dimerization domains allow reversible growth inhibition by p16. Nat Genet 20 , 389-393.


RetroTet 2 (activator only)
RetroTet 2 is a bipartite system of RetroTet RTAb(-) or RetroTet RTAb(+) with a Hermes regulatable vector such as Hermes HRSpuro-GFP or HRSpuro-GUS. This activator-only system permits tetracycline regulated expression of the gene of interest using a transactivator that binds either in the absence (RetroTet RTAb(-)) or presence (RetroTet RTAb(+)) of tetracycline.

The use of these vectors should be cited as follows: Kringstein, A. M., Rossi, F. M., Hofmann, A., & Blau, H. M. (1998). Graded transcriptional response to different concentrations of a single transactivator. Proc Natl Acad Sci U S A 95, 13670-13675.


Name


Plasmids


Function


RetroTet ART





RetroTet RTAb(+)
RetroTet RTRg(-)cd8
RetroTet RTRg(-)gfp
Hermes HRSpuro-GUS
Hermes HRSpuro-gfp

Activator
Repressor
Repressor
Regulatable vector
Regulatable vector

RetroTet 2
(activator only)



RetroTet RTAb(-)
RetroTet RTAb(+)
Hermes HRIgfp

Activator
Activator
Regulatable vector


RetroTet Transactivator/Transrepressor Constructs Overview:
The RetroTet constructs express either a transactivator (RTA) or transrepressor (RTR) for use in regulating gene expression from one of the Hermes vectors. The portion of the transactivator or transrepressor that is derived from the bacterial tetracycline repressor (tetR) is either of the B class (b), or is a hybrid of the B and G classes (g). The b and g molecules cannot heterodimerize. The tetR portion responds to tetracycline (tet) and its analogs (e.g. doxycycline) either by binding DNA in the absence of tet, designated (-), or in the presence of tet, designated (+). The transactivator domain is from VP16; the transrepressor domain is from the KRAB domain of Kox-1.

RetroTet RTAb(-) : Plasmid Map | Sequence
Transactivator (A) that binds DNA (tet operator) and activates transcription in the ABSENCE (-) of tetracycline and its analogs. Utilizes the wild-type tetR with the B-class (b) dimerization domain that is fused to the VP16 transcriptional activation domain (= tTA of Bujard). Expressed in a constitutively active high titre MFG retroviral backbone. Cannot be used in combination with RetroTet RTRg(-) transrepressor vectors. Kringstein et al. 1998, PNAS 95 , 13670-13675.

RetroTet RTAb(+) : Plasmid Map | Sequence
Transactivator (A) that binds DNA (tet operator) and activates transcription in the PRESENCE (+) of tetracycline and its analogs. Utilizes a mutated tetR moiety with the B-class (b) dimerization domain that is fused to the VP16 transcriptional activation domain (= rtTA of Bujard). Expressed in a constitutively active high titre MFG retroviral backbone. Can be used in combination with RetroTet RTRg(-) transrepressor vectors (RetroTet ART system). Kringstein et al. 1998, PNAS 95, 13670-13675; Spicher et al. 1998, MCB 18, 7371-7382; Rossi et al. 1998, Nature Genetics 20 , 389-393.


RetroTet RTRg(-) :
Transrepressor (R) that binds DNA (tet operator) and represses transcription in the ABSENCE (-) of tetracycline and its analogs. Utilizes a tetR with a hybrid B- and G-class (g) dimerization domain that is fused to the KRAB transcriptional repression domain of Kox1. The class-switching of the dimerization domain abrogates the formation of non-productive heterodimers when used in conjunction with RetroTet RTAb(+) transactivator vectors (RetroTet ART system). Expressed in a constitutively active high titre MFG retroviral backbone. Rossi et al. 1998, Nature Genetics 20 , 389-393.


RetroTet RTRg(-)cd8 : Plasmid Map | Sequence
Transrepressor (R) that binds DNA (tet operator) and represses transcription in the ABSENCE (-) of tetracycline and its analogs. Utilizes a tetR with a hybrid B- and G-class (g) dimerization domain that is fused to the KRAB transcriptional repression domain of Kox1. The class-switching of the dimerization domain abrogates the formation of non-productive heterodimers when used in conjunction with RetroTet RTAb(+) transactivator vectors (RetroTet ART system). Expressed in a constitutively active high titre MFG-derived retroviral backbone, which also expresses the marker CD8 (Lyt2) downstream of the transrepressor via a bicistronic transcript containing an encephalomyocarditis (EMCV) derived internal ribosome entry site (IRES). Rossi et al. 1998, Nature Genetics 20 , 389-393.


RetroTet RTRg(-)gfp : Plasmid Map | Sequence
Transrepressor (R) that binds DNA (tet operator) and represses transcription in the ABSENCE (-) of tetracycline and its analogs. Utilizes a tetR with a hybrid B- and G-class (g) dimerization domain that is fused to the KRAB transcriptional repression domain of Kox1. The class-switching of the dimerization domain abrogates the formation of non-productive heterodimers when used in conjunction with RetroTet RTAb(+) transactivator vectors (RetroTet ART system). Expressed in a constitutively active high titre MFG-derived retroviral backbone, which also expresses the marker GFP (green fluorescent protein, enhanced or EGFP variety) downstream of the transrepressor via a bicistronic transcript containing an encephalomyocarditis (EMCV) derived internal ribosome entry site (IRES). Rossi et al. 1998, Nature Genetics 20 , 389-393.


Hermes Regulatable Vectors Overview:
Hermes regulatable (HR) vectors, named for the "messenger of the gods", allow the regulated expression of a gene of interest when used in conjunction with a tetracycline regulated transactivator or transrepressor. A transactivator and a transrepressor from different classes can be used simultaneously to regulate expression from the Hermes vector over a greater range than is possible with either alone. The vectors most useful for cloning new genes of interest are Hermes HRIgfp and Hermes HRSpuro-GUS.


Hermes HRIgfp : Plasmid Map | Sequence
Tetracycline-regulatable expression vector containing a "superminimal" CMV promoter (position -53 to +2) fused to heptamerized tet operator (O7) sequences upstream of a polylinker. A gene of interest can be cloned into the polylinker, and is expressed from a bicistronic transcript which also contains an encephalomyocarditis virus (EMCV) derived internal ribosome entry site (IRES, I) followed by the marker EGFP (enhanced green fluorescent protein). Transcription of both the gene of interest and the marker can be regulated by tetracycline and are in sense orientation to the viral LTR. The vector backbone is an MFG-derived self-inactivating (SIN) retrovirus, which prevents transcription from the constitutive viral LTR. Similar to vector described in Kringstein et al. 1998, PNAS 95 , 13670-13675., but lacks human growth hormone gene.


Hermes HRS vectors
Hermes S vectors contain a tetracycline-regulatable gene of interest downstream of the tet operator sequences and CMV promoter, all in the antisense orientation to the viral LTR. The vector also contains a constitutive SV40 enhancer driven cassette which expresses a marker and is oriented in the sense orientation to the viral LTR. The "S" in HERMES S designates the use of the SV40 driven constitutive marker oriented opposite to the regulatable gene of interest. The SV40 enhancer is followed by a marker (shown in lower case), typically green fluorescent protein (gfp), CD8/Lyt2 (cd8), or puromycin resistance (puro). The gene of interest, if present, is set off by a hyphen and shown in upper case in the vector name.


Hermes HRSpuro-gfp : no map is available but is similar to HRSpuro-GUS with gfp replacing GUS
Expression vector containing two distinct transcriptional cassettes. A tetracycline regulatable cassette oriented antisense to the viral LTR contains a minimal CMV promoter (position -53 to +75) fused to heptamerized tet operator (O7) sequences upstream of the human codon-optimized blue-excitable green fluorescent protein (GFP-Bex1), followed by a bovine growth hormone poly-A sequence. The second constitutively expressed cassette is oriented sense to the viral LTR and contains a constitutively expressed marker gene encoding resistance to the antibiotic puromycin (puro), driven by the SV40 promoter. The vector backbone is an MFG-derived self-inactivating (SIN) retrovirus , which prevents transcription from the constitutive viral LTR. Rossi et al. 1998, Nature Genetics 20 , 389-393.


Hermes HRSpuro-GUS : Plasmid Map | Sequence
Expression vector containing two distinct transcriptional cassettes. A tetracycline regulatable cassette oriented antisense to the viral LTR contains a minimal CMV promoter (position -53 to +75) fused to heptamerized tet operator (O7) sequences upstream of a 5' polylinker preceding the beta-glucoronidase gene (GUS) followed by a 3' polylinker and by a bovine growth hormone poly-A sequence. The second constitutively expressed cassette is oriented sense to the viral LTR and contains a constitutively expressed marker gene encoding resistance to the antibiotic puromycin (puro), driven by the SV40 promoter. The vector backbone is an MFG-derived self-inactivating (SIN) retrovirus , which prevents transcription from the constitutive viral LTR. The 5' and 3' polylinkers make this a useful vector for cloning 5' and 3' untranslated sequences that have a regulatory role (riboregulators). Spicher et al. 1998, MCB 18 , 7371-7382.