| Regulatable Retroviral Vector Systems | |
| System to Knockdown Gene Expression | Systems for Monitoring Protein-Protein Interactions |
| Antibodies | Cell Cultures |
Regulatable Retroviral Vector Systems
These systems are recommended for cell culture or animals "short-term".
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.
Five vectors constitute the RetroTet ART System:
| Activator | Repressor | Regulatable Vector |
| RetroTet RTAb(+) | RetroTet RTRg(-)cd8 | Hermes HRSpuro-GUS |
| RetroTet RTRg(-)gfp | Hermes HRSpuro-gfp |
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.
Three vectors constitute the RetroTet 2 system:
| Activator | Regulatable Vector |
| RetroTet RTAb(-) | Hermes HRIgfp |
| RetroTet RTAb(+) |
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.
Vector Overview
1) 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 |
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RetroTet RTAb(-) : Plasmid Map | Sequence | top 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. |
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RetroTet RTAb(+) : Plasmid Map | Sequence | top 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. |
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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. |
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RetroTet RTRg(-)cd8 : Plasmid Map | Sequence | top 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. |
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RetroTet RTRg(-)gfp : Plasmid Map | Sequence | top 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. |
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2) 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. |
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Hermes HRIgfp : Plasmid Map | Sequence | top 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. |
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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. |
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Hermes HRSpuro-gfp : top 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. |
no map is available but is similar to HRSpuro-GUS with gfp replacing GUS |
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Hermes HRSpuro-GUS : Plasmid Map | Sequence | top 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. |
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Systems for Monitoring Protein-Protein Interactions
Beta-Galactatosidase Complementation
This is an approach for monitoring protein-protein interactions within intact eukaryotic cells that can increase our understanding of the regulatory circuitry that controls the proliferation and differentiation of cells and how these processes go awry in disease states such as cancer. Chimeric proteins composed of proteins of interest fused to complementing beta-galactosidase (beta-gal) deletion mutants permit a novel analysis of protein complexes within cells. In this approach, the beta-gal activity resulting from the forced interaction of non-functional weakly complementing beta-gal peptides (delta alpha and delta omega) serves as a measure of the extent of interaction of the non-beta-gal portions of the chimeras. This approach may prove to be a valuable adjunct to in vitro immunoprecipitation and crosslinking methods, and in vivo yeast two-hybrid and fluorescence energy transfer systems. It may also allow a direct assessment of specific protein dimerization interactions in a biologically relevant context, localized in the cell compartments in which they occur, and in the milieu of competing proteins.
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| pWZL Delta-alpha Neo | Plasmid Map | Sequence | |
| pWZL Delta-omega Hygro | Plasmid Map | Sequence |
The use of these vectors should be cited as follows: Mohler, W. A., & Blau, H. M. (1996). Gene expression and cell fusion analyzed by lacZ complementation in mammalian cells. Proc Natl Acad Sci U S A 93 , 12423-7.
| FACS (cell sorting) | Protocol | |
| Chemiluminescence | Protocol | |
| Histology (x-gal and Fluor-x-gal) | Protocol |
Beta-Lactamase Complementation
We have defined inactive alpha and omega fragments of beta-lactamase that can complement to form a functional enzyme in both bacteria and mammalian cells, serving as a readout for the interaction of proteins fused to the fragments. Critical to this advance was the identification of a tripeptide, Asn-Gly-Arg, which when juxtaposed at the carboxyl terminus of the alpha fragment increased complemented enzyme activity by up to 4 orders of magnitude. Beta-lactamase is well suited to monitoring constitutive and inducible protein interactions because it is small (29 kDa), monomeric, and assayable with a fluorescent cell-permeable substrate. The negligible background, the magnitude of induced signal caused by enzymatic amplification, and detection of signal within minutes are unparalleled in mammalian protein interaction detection systems published to date.
| Mammalian Expression Vectors | Plasmid Map | |
| pWZL-alpha-gs-NEO | Sequence | |
| pWZL-W_GS-HYGRO | Sequence | |
| Bacterial Expression Vectors | Plasmid Map | |
| Fos-GS-W | Sequence | |
| A-GS-Jun | Sequence |
The use of these vectors should be cited as follows: Wehrman, T.S., Kleaveland, B.M., Her, J-H., Balint, R. and Blau, H.M. (2002) Protein-protein interactions monitored in mammalian cells via complementation of b-lactamase enzyme fragments, Proc. Natl. Acad. Sci. USA 99 , 3469-3474.
| Staining | Protocol |
Restriction Enzyme-Generated siRNA (REGS) vectors
REGS products are cloned directly in vREGS to facilitate the construction of retroviral siRNA constructs
| vREGS | Sequence |
The use of these vectors should be cited as follows: Sen, G., Wehrman, T.S., Meyers, J.W. and Blau, H.M. (2004) Restriction enzyme-generated siRNA (REGS) vectors and libraries, Nature Genetics 36:2 , 183-189.
Cell Cultures
| C2 Cell Line | C2 mouse myoblasts are available from the American Type Culture Collection (ATCC) . The product number is CRL-1772. | Protocol |
| Human Primary Myoblasts | We do not have any permanent human muscle cell lines. We only have primary skeletal myoblast cultures. Since each of these cultures is unique and irreplaceable, we generally do not send them to other labs - sorry! | Protocol |
| Mouse Primary Myoblasts | Protocol | |
| Hoechst Staining | Protocol | |
| Heterokaryons | Protocol |
Antibodies
Anti-Myosin Heavy Chain Antibodies
Most of the hybridomas for the anti-myosin heavy chain (MyHC) antibodies that originated in this lab and were used in our publications are available from the American Type Culture Collection (ATCC) or from the Developmental Studies Hybridoma Bank (DSHB) . The DSHB can provide hybridoma supernatants, purified IgG, and ascites if you do not want the cells. If you are looking for one of our published antibodies and it is not available from these sources, it is probably because we think that one of the available antibodies has similar activity and is in some way superior.
A4.1025 |
human and rodent, all fiber types |
not available |
93199 |
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A4.74 |
human fast and rodent fast IIa, IIx,d |
CRL-2041 |
93201 |
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N2.261 |
human and rodent slow and fast IIa |
CRL-2047 |
93206 |
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F1.652 |
human and rodent embryonic (not adult) |
CRL-2039 |
93204 |
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N3.36 |
human and rodent neonatal and adult fast |
CRL-2042 |
93207 |
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N1.551 |
rodent fast IIa, IIx |
CRL-2043 |
93205 |
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A4.840 |
human and rodent slow |
CRL-2040 |
93202 |
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A4.951 |
human and rodent slow |
CRL-2046 |
93203 |
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A4.1519 |
human and rat adult, some embryonic |
not available |
93200 |
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* In many of our publications using these antibodies, the name of the antibody may appear slightly different. In particular, the number and letter preceding the period in the antibody name may be reversed, e.g. 4A.1025 instead of A4.1025. However, the numbers following the period in the name should be unchanged.
Anti-Human N-CAM Antibody (5.1H11)
The anti-human neural cell adhesion molecule (N-CAM) antibody 5.1H11 originated in the lab of Frank Walsh, and has been employed in several of our publications. The hybridoma that produces this antibody is available from the Developmental Studies Hybridoma Bank (DSHB) , not from the ATCC.
This antibody is useful for purifying human primary myoblasts.