a vector set for plasmid -based rnai studies in mammalian cells
TRANSCRIPT
A Vector Set For Plasmid-Based RNAi Studies in Mammalian Cells Version 1.0, May 2004
Iain D.C. Fraser Alliance for Cell Signaling, Molecular Biology Laboratory California Institute of Technology, Pasadena, CA
Introduction RNA interference (RNAi) has recently emerged as a powerful experimental tool
in mammalian cell biology. Double-stranded RNA (dsRNA)-induced gene silencing was originally discovered as a mechanism for regulating expression of endogenous genes in C. elegans (1), although a similar phenomenon, termed cosuppression, had been demonstrated in plants (2). Studies of the RNAi mechanism demonstrated that the degradation of the target mRNA was mediated by short fragments of dsRNA (3, 4), which were shown to be derived from the longer dsRNA template through the action of the ribonuclease dicer (5). These short interfering RNAs (siRNAs), when exogenously introduced, can enter the RNAi pathway downstream of dicer and efficiently reduce expression of their target gene. The RNAi pathway was not initially considered a useful experimental tool in mammalian cells due to interferon responses to the dsRNA trigger (6). However, Tuschl and colleagues hypothesized that this approach could be applied to mammalian cells if introduction of the shorter siRNAs could avoid activation of the interferon pathway (7). They confirmed their hypothesis in several mammalian cells lines (7), and RNAi has now been shown to be a valuable experimental tool in a variety of biological systems (8).
The direct transfection of chemically synthesized siRNA duplexes into mammalian cells, as originally demonstrated by the Tuschl lab, is currently the most popular approach to RNAi. However, the applicability of this technique is dependent on the transfectability of the model cell system, and since the presence of the siRNA in the cell is transient, longer term experiments are more difficult. This issue has been addressed by the development of approaches that permit the expression of siRNAs from DNA-based plasmid vectors. Several groups have shown that siRNAs can be transcribed as stem-loop hairpin structures under the control of RNA polymerase III (pol III) promoters (9-13). Figure 1 shows a schematic of how such a short hairpin RNA (shRNA) can be transcribed from the promoter for the RNA component of RNase P, H1. Similar expression cassettes have also been developed using the promoter for the small nuclear RNase, U6.
A Vector Set for Plasmid-Based RNAi Studies in Mammalian Cells AfCS Reports, Version 1.0, May 2004 Page 2
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Fig. 1. Expression of an shRNA from a pol III expression cassette. N19 represents a 19 nucleotide sequence homologous to the target gene of interest, and N’19 represents the complementary sequence of the antisense strand. The cassette is designed such that the first base of the N19 sequence is positioned at the transcriptional start site of the H1 promoter. The termination signal for transcription is a sequence of four thymidines.
____________________________________________________________ The pol III promoter-based system described in this document allows for the expression of siRNAs from either mammalian expression vectors or viruses, thus permitting the transduction of difficult-to-transfect cell lines and primary cell cultures. This system also allows for the creation of stable cell lines depleted in a specific target gene.
H1 pr. N19-TT-loop-N’19 TTTT
+1
UU
UU
Transcription
5’-
UU
N’19UU
5’-
-5’
Processing
Short hairpin RNA
siRNA duplex
N’19
N19
N19
TerminatorPromoter shRNA Cassette
H1 pr. N19-TT-loop-N’19 TTTT
+1
UU
UU
Transcription
5’-
UU
N’19UU
5’-
-5’
Processing
Short hairpin RNA
siRNA duplex
N’19
N19
N19
TerminatorPromoter shRNA Cassette
A Vector Set for Plasmid-Based RNAi Studies in Mammalian Cells AfCS Reports, Version 1.0, May 2004 Page 3
Based on the architecture of the expression cassette shown in Fig. 1, several groups have developed vector systems that permit the insertion of an shRNA cassette between a promoter and terminator sequence using restriction enzyme sites adjacent to these transcriptional elements (Fig. 2). ____________________________________________________________
Fig. 2. Creation of a plasmid vector for shRNA expression. The shRNA cassette is created by the annealing of two complementary oligonucleotides containing the desired sequence.
____________________________________________________________
Using this approach, the vector backbone can be varied to permit expression of the shRNA in a variety of contexts. Many companies now sell vectors for shRNA expression based on this principal. Although powerful, this approach requires laborious cloning and sequencing steps for every vector into which a given shRNA is cloned. This document describes a more flexible vector system for shRNA expression.
TerminatorPromoter
N19-TT-loop-N’19
H1 pr. TTTT
Restriction Enzyme Site 1
Restriction Enzyme Site 2
shRNA Cassette
Vector Backbone
TerminatorPromoter
N19-TT-loop-N’19
H1 pr. TTTT
Restriction Enzyme Site 1
Restriction Enzyme Site 2
shRNA Cassette
Vector Backbone
A Vector Set for Plasmid-Based RNAi Studies in Mammalian Cells AfCS Reports, Version 1.0, May 2004 Page 4
Gateway-Based Vectors for shRNA Cloning Background
Using the Gateway cloning technology (Invitrogen), we have developed a more flexible vector system that reduces time spent subcloning shRNAs into multiple expression vectors. Detailed background on Gateway cloning is available at the Invitrogen Web site (http://www.invitrogen.com). Briefly, the system uses site-specific recombination instead of traditional restriction enzymes and ligase for subcloning recombinant DNA. Although this system was developed primarily for the subcloning of cDNAs, it can be readily applied to the subcloning of the pol III-shRNA cassettes shown in Fig.1 and Fig. 2.
The first step in the use of the Gateway system is to clone the DNA sequence of interest into a so-called entry vector. The basic property of an entry vector is that it contains the attL1 and attL2 recombination sites at either side of the DNA sequence that will eventually be shuttled into different expression systems. We have created entry vectors containing either a human or mouse H1 promoter (pEN_hH1c and pEN_mH1c, respectively). Figure 3 shows a schematic detailing the shRNA cloning process and the restriction enzyme sites used for these entry vectors. ____________________________________________________________
Fig. 3. Ligation of an shRNA cassette into either the pEN_hH1c or pEN_mH1c entry vectors.
____________________________________________________________
Note that full vector maps for the pEN_hH1c and pEN_mH1c plasmids are provided in Appendix 1. Table 1 provides names, ATCC IDs, AfCS bar codes, and relevant vector details for each of these vectors.
attL1 ccdB attL2
BamH1 Xho1
pEN_hH1cor
pEN_mH1c
+
H1 pr.
N19-TT-loop-N’19
TTTT
TerminatorPromoter
shRNA Cassette
attL1 attL2
BamH1 Xho1
H1 pr. TTTTN19-TT-loop-N’19
attL1 ccdB attL2
BamH1 Xho1
pEN_hH1cor
pEN_mH1c
+
H1 pr.
N19-TT-loop-N’19
TTTT
TerminatorPromoter
shRNA Cassette
attL1 attL2
BamH1 Xho1
H1 pr. TTTTN19-TT-loop-N’19
A Vector Set for Plasmid-Based RNAi Studies in Mammalian Cells AfCS Reports, Version 1.0, May 2004 Page 5
Table 1. Details of entry vectors for shRNA cloning.
Vector name ATCC ID AfCS bar code Vector details
pEN_hH1c 10326368 P05EENHH1CXG shRNA cloning and expression; Human H1 promoter; Entry vector backbone; +ccdB in parent
pEN_mH1c 10326369 P06EENMH1CXG shRNA cloning and expression; Mouse H1 promoter; Entry vector backbone; +ccdB in parent
The schematic in Fig. 3 shows an element located between the BamH1 and Xho1 enzymes sites in the pEN_H1 vectors labeled ccdB. This element is excised during the cloning of the shRNA cassette. The ccdB gene product is toxic to commonly used strains of E. coli, so the presence of this gene in the pEN_H1 parent vectors means that when the products of the shRNA ligation reaction are used to transform a common E. coli strain, there can be no background colonies from uncut or single-cut parent. We have found that this approach significantly increases the percentage of transformant s containing the desired shRNA sequence.
Design and Synthesis of an shRNA Sequence against a Target Gene Selection Criteria for Gene-Specific shRNA Sequences
Until recently, the dogma in the RNAi field was that there was no reliable method of rationally designing effective siRNA target sequences for any given mRNA. Our general approach in designing shRNAs for expression in the pEN_H1 vectors has been to choose four sequences, spaced throughout the mRNA of each gene we want to target, and then to test each shRNA for efficacy. Some general rules for sequence selection are as follows:
1. Choose a gene-specific sequence of 19 to 21 nucleotides. 2. The GC content of the sequence should be between 45% and 55%. 3. The Tm of the sequence should be between 45 °C and 65 °C. 4. Avoid runs of two or more As at beginning of sequence (causes premature
termination of transcription; see Fig.4). 5. Avoid runs of two or more Ts at end of sequence (causes premature termination
of transcription; see Fig.4). 6. Run the sequence through a BLAST search to ensure specificity for the target
gene. More recently, several groups have developed algorithms that permit more rational
siRNA design. Dharmacon (http://www.dharmacon.com) has developed a process termed SMARTpool, which uses over 30 criteria that improve the likelihood that a selected sequence will represent an effective siRNA. Dharmacon also offers a free basic version of their algorithm (using a handful of their criteria) on their Web site in their siDESIGN center. This does not provide the high success rate of their custom SMARTpool sequence
A Vector Set for Plasmid-Based RNAi Studies in Mammalian Cells AfCS Reports, Version 1.0, May 2004 Page 6
selection, but it does provide a resource that is an improvement over random selection. Further information on the current status of research in the RNAi field and shRNA design is also available from the following Web sites: http://www.cshl.edu/public/SCIENCE/hannon.html http://www.rockefeller.edu/labheads/tuschl/sirna.html
Once a sequence from a given mRNA has been selected for shRNA expression, it must be incorporated into a cassette for cloning into one of the pEN_H1 plasmids. Design of the shRNA Cassette
For both pEN_hH1c and pEN_mH1c, the shRNA cassette is cloned into BamH1 and Xho1 restriction enzyme sites. The shRNA cassette is designed such that, after annealing of two complementary oligos, the cassette has ssDNA sequences at both ends that are compatible with the BamH1 and Xho1 restriction sites. Figure 4 demonstrates this with a template for cassette design and an example for an shRNA directed against the G alpha i2 gene. ____________________________________________________________
Template 1 2 5’-gatcccc>>>>>>>>>>>>>>>>>>>ttcaagaga<<<<<<<<<<<<<<<<<<<tttttc-3’ 3’-ggg<<<<<<<<<<<<<<<<<<<aagttctct>>>>>>>>>>>>>>>>>>>aaaaagagct-5’ G alpha i2 example Sequence selected from G alpha i2 mRNA: GCACAGAGTGACTACATCC 5’-GATCCCCGCACAGAGTGACTACATCCTTCAAGAGAGGATGTAGTCACTCTGTGCTTTTTC-3’ 3’-GGGCGTGTCTCACTGATGTAGGAAGTTCTCTCCTACATCAGTGAGACACGAAAAAGAGCT-5’ Gi2 sense oligo GATCCCCGCACAGAGTGACTACATCCTTCAAGAGAGGATGTAGTCACTCTGTGCTTTTTC Gi2 antisense oligo TCGAGAAAAAGCACAGAGTGACTACATCCTCTCTTGAAGGATGTAGTCACTCTGTGCGGG
Fig. 4. Template for shRNA cassette destined for the pEN_hH1c or pEN_mH1c entry vector. For the first oligonucleotide (top), the 19 nucleotide gene-specific sequence should replace the > characters in the first pink region (1), and the reversed and complementary antisense sequence should replace the < characters in the second pink region (2). For the second oligonucleotide (bottom), the first four bases in red at the 5’ end provide the Xho1 compatible sequence, while the remaining sequence is complementary to the first oligonucleotide.
____________________________________________________________
A Vector Set for Plasmid-Based RNAi Studies in Mammalian Cells AfCS Reports, Version 1.0, May 2004 Page 7
Cloning and Testing shRNAs Cloning of shRNA Linkers into the pEN_hH1c or pEN_mH1c Vector
Upon synthesis of sense and antisense oligos for an shRNA cassette, the oligos must be annealed to form a linker for ligation into one of the pEN_H1 entry vectors shown in Table 1. This procedure is detailed in the AfCS protocol PP00000230. Each candidate shRNA clone must be fully sequence verified to ensure that it retains 100% homology to the target gene. Testing of shRNA Linkers in the pEN_H1 Vectors
The pEN_H1 vectors contain all the elements necessary for shRNA expression in mammalian cells. Once an shRNA has been sequence validated, the resulting construct can be transfected into a mammalian cell by established methodology. The effect of the shRNA on target gene expression can then be assessed by either RT-PCR or Western blot.
Note that in cell lines that are difficult to transfect, it may be difficult to assess shRNA efficacy due to low transfection efficiency of the plasmid. We find that for shRNA testing, it is useful if a full- length cDNA construct is available for the target gene. In these circumstances, we generally cotransfect HEK293 cells with the shRNA-expressing plasmid and another plasmid expressing a GFP-tagged fusion of the target gene. shRNA efficacy is then assessed by Western blot using an antibody against GFP.
In cell lines that transfect to a high efficiency, validated shRNAs transiently expressed from the pEN_H1 vectors may be sufficient for assessing the consequences of target gene knockdown in many cell-based assays. However, in more difficult-to-transfect cell lines and primary cells, an alternative transduction approach is necessary to ensure that an shRNA is expressed throughout a cell population. One such approach would be to express an shRNA from a mammalian expression vector containing a drug resistance gene that would permit selection of a stable cell line after transfection. Another possibility would be to subclone the shRNA cassette into a viral vector, allowing more efficient infection of cells with virus, followed by the subsequent selection of a stable line in cases where the viral vector carries a drug resistance gene.
Use of a Gateway entry vector for shRNA cloning opens up the possibility of simple subcloning of H1 promoter-shRNA cassettes into multiple expression systems.
A Vector Set for Plasmid-Based RNAi Studies in Mammalian Cells AfCS Reports, Version 1.0, May 2004 Page 8
Subcloning of Pol III-shRNA Cassettes via the LR Recombination Reaction
As mentioned above, the Gateway cloning system can reduce time spent subcloning shRNAs into different expression systems. We have created a wide range of gateway-compatible expression vectors, allowing users to choose the shRNA expression method best suited to their needs.
Figure 3 showed that after an shRNA cassette is ligated into one of the pEN_H1 vectors, the H1 promoter-shRNA-transcription terminator elements are flanked by attL recombination sites. Consequently, if such an attL-containing vector is combined with an attR-containing vector in an LR recombination reaction, the H1 promoter-shRNA-transcription terminator elements will be moved to the attR-containing vector.
Figure 5 shows a schematic for this LR recombination reaction. The reagents and experimental protocol for an LR reaction are available from Invitrogen. ____________________________________________________________
Fig. 5. LR recombination reaction resulting in the movement of an shRNA expression cassette from a pEN_H1 vector to one of the destination vectors developed for shRNA expression. ____________________________________________________________
It should be noted from Fig. 5 that for an entry and destination vector to be compatible in an LR reaction, they must have different bacterial selection markers to allow for appropriate selection of the resulting expression clones. For this reason, we
pEN_hH1-shRNAor
pEN_mH1-shRNA
ccdBattR1 attR2
H1 pr. TTTTshRNAattB1 attB2
H1 pr. TTTTshRNAattL1 attL2
+
Any of destination vectors for shRNA
expression from Table 2
+By-product
shRNA-expressing viral or mammalian
expression vector
LR recombination
Amp or Kan
Gen
Amp or Kan
pEN_hH1-shRNAor
pEN_mH1-shRNA
ccdBattR1 attR2
H1 pr. TTTTshRNAattB1 attB2
H1 pr. TTTTshRNAattL1 attL2
+
Any of destination vectors for shRNA
expression from Table 2
+By-product
shRNA-expressing viral or mammalian
expression vector
LR recombination
Amp or Kan
Gen
Amp or Kan
A Vector Set for Plasmid-Based RNAi Studies in Mammalian Cells AfCS Reports, Version 1.0, May 2004 Page 9
developed the pEN_hH1c and pEN_mH1c entry vectors as gentamicin-resistant plasmids (see maps in Appendix 1). This allows the shRNA expression cassettes to be moved to expression vectors that are either ampicillin- or kanamycin-resistant. Any expression vector containing the attR1-ccdB-attR2 elements at an appropriate location for an shRNA cassette is a compatible destination vector for shRNA expression. We have created a range of mammalian expression and viral expression vectors for this purpose. Expression Systems for shRNAs
Maps of the available destination vectors we have constructed for shRNA expression are provided in Appendix 2. Table 2 provides names, ATCC, AfCS bar codes, vector types, and relevant details for each of these vectors.
Note that the presence of the attR1-ccdB-attR2 elements (which total almost 2kb) in each of these destination vectors makes them inappropriate as empty vector controls for experimental work. Therefore, in Table 2, we also provide details of control vectors lacking the attR1-ccdB-attR2 elements present in the corresponding destination vector. Maps for these control vectors are provided in Appendix 3. For the three mammalian expression vectors at the bottom of Table 2, appropriate empty vector controls are available from commercial sources. Table 2. Details of vector systems for shRNA expression. Name ATCC ID AfCS bar code Vector type Features coexpressed with shRNA pDSL_hpUGIP 10326373 L11DDLUGIPXA Lentiviral Ubi-c promoter-driven GFP -IRES-Puromycin
pL_UGIP 10326372 L10GLUGIP1XA Lentiviral Ubi-c promoter-driven GFP -IRES-Puromycin (control for pDSL_hpUGIP)
pDSL_hpUGIH 10326379 L24DDLUGIHXA Lentiviral Ubi-c promoter-driven GFP -IRES-Hygromycin
pL_UGIH 10326378 L23GLUGIH1XA Lentiviral Ubi-c promoter-driven GFP -IRES-Hygromycin (control for pDSL_hpUGIH)
pDSL_hpUC 10326377 L20DDLHPUCXA Lentiviral Ubi-c promoter-driven delta CD4
pL_UC 10326376 L19GLUC001XA Lentiviral Ubi-c promoter-driven delta CD4 (control for pDSL_hpUC)
pDSL_hpUP 10326375 L12DDLHPUPXA Lentiviral Ubi-c promoter-driven Puromycin
pL_UP 10326374 L17GLUP001XA Lentiviral Ubi-c promoter-driven Puromycin (control for pDSL_hpUP )
pDSL_hpUG 10326371 L06DDLHPUGXA Lentiviral Ubi-c promoter-driven GFP
pL_UG 10326370 L01GLUG001XA Lentiviral Ubi-c promoter-driven GFP (control for pDSL_hpUG)
pDS_CXGhp 10326381 B03D0CXGHPXA Retroviral CMV promoter-driven GFP and 5'LTR-driven Puromycin
pCXG 10326380 M0001CXG000A Retroviral CMV promoter-driven GFP and 5'LTR-driven Puromycin (control for pDS_CXGhp)
pDS_hpSC 10326382 B88DDSHPSCXA Mammalian SV40 promoter-driven delta CD4
pDS_hpCG 10326383 B94DDSHPCGXK Mammalian CMV promoter-driven GFP and SV40-driven Neomycin
pDS_hpEY 10326384 B95DDSHPEYXK Mammalian EF1 promoter-driven YFP and SV40-driven Neomycin
All of the vectors detailed in Tables 1 and 2 are now available from the American Type Culture Collection (http://www.atcc.org/). When ordering, specific vectors should be referenced by their ATCC ID.
A Vector Set for Plasmid-Based RNAi Studies in Mammalian Cells AfCS Reports, Version 1.0, May 2004 Page 10
References 1. Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, and Mello CC. (1998)
Nature 391(6669), 806-811. PMID: 9486653. 2. Matzke MA, Aufsatz W, Kanno T, Mette MF, and Matzke AJ. (2002)
Adv. Genet. 46, 235-275. PMID: 11931226. 3. Hamilton AJ and Baulcombe DC. (1999) Science 286(5441), 950-952.
PMID: 10542148. 4. Zamore PD, Tuschl T, Sharp PA, and Bartel DP. (2000) Cell 101(1), 25-33.
PMID: 10778853. 5. Hammond SM, Bernstein E, Beach D, and Hannon GJ. (2000) Nature 404(6775),
293-296. PMID: 10749213. 6. Samuel CE. (2001) Clin. Microbiol. Rev. 14(4), 778-809. PMID: 11585785. 7. Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, and Tuschl T. (2001) Nature 411(6836), 494-498. PMID: 11373684. 8. Hannon GJ. (2002) Nature 418(6894), 244-251. PMID: 12110901. 9. Paddison PJ, Caudy AA, Bernstein E, Hannon GJ, and Conklin DS. (2002)
Genes Dev. 16(8), 948-958. PMID: 11959843. 10. Brummelkamp TR, Bernards R, and Agami R. (2002) Science 296(5567), 550-
553. PMID: 11910072. 11. Paul CP, Good PD, Winer I, and Engelke DR. (2002) Nat. Biotechnol. 20(5), 505-
508. PMID: 11981566. 12. Sui G, Soohoo C, Affar el B, et al. (2002) Proc. Natl. Acad. Sci. U. S. A. 99(8),
5515-5520. PMID: 11960009. 13. Yu JY, DeRuiter SL, and Turner DL. (2002) Proc. Natl. Acad. Sci. U. S. A. 99(9),
6047-6052. PMID: 11972060.
A Vector Set for Plasmid-Based RNAi Studies in Mammalian Cells AfCS Reports, Version 1.0, May 2004 Page 11
Appendix 1: Vector Maps for pEN_H1 Entry Vectors P05EENHH1CXG Full sequence for this plasmid is available at the AfCS data center (http://www.signaling-Gateway.org/data/Data.html). Follow the Plasmid Database: Web interface link in the Resources section and use the above bar code to retrieve plasmid data.
XmnI
XmaI
XhoI
Tth111I
SmaI
SfoISacII
PvuII
PvuI
PstI
PflMI
PflFI
PaeR7I
NruI
NheI
NcoI
NarI
KpnI
KasI
HincII
EcoO109I
EcoNI
DrdI
DraIII
BtrI
BstZ17I
BstBI
BstAPI
BssSI
BseRIBsaAI
BglII
BglI
BciVI
BamHI
BaeI-bBaeI-a
AvaII
AlwNI
AflII
AclI
AccI
Acc65I
0.0kb
0.5kb
1 .0kb
1.5kb
2.0kb
2.5kb
3.0k
b
3.5k
b
attL1hH1
ccdB
attL2o r
i
Gen
pEN_hH1c4114 bp
A Vector Set for Plasmid-Based RNAi Studies in Mammalian Cells AfCS Reports, Version 1.0, May 2004 Page 12
P06EENMH1CXG Full sequence for this plasmid is available at the AfCS data center (http://www.signaling-Gateway.org/data/Data.html). Follow the Plasmid Database: Web interface link in the Resources section and use the above bar code to retrieve plasmid data.
XmnI
XmaI
XhoI
StyI
SspI
SmaI
SacII
PvuII
PvuI
PstI
PspOMI
PflMI
PaeR7I
NruI
NheIKpnI
HincII
EcoO109I
EcoNI
DrdI
DraIII
BtrI
BstZ17IBssSIBssHII
BseRIBsaAI
BglII
BglI
BciVI
BbsI
BbsI BanI
BamHI
BaeI-bBaeI-a
AvaII
ApaI
AlwNI
AflII
AclI Acc65I
0.0kb
0.5kb
1. 0kb
1.5kb
2.0kb
2.5kb
3.0k
b
3.5k
b
attL1mH1
ccdB
attL2o r
i
Gen
pEN_mH1c4060 bp
A Vector Set for Plasmid-Based RNAi Studies in Mammalian Cells AfCS Reports, Version 1.0, May 2004 Page 13
Appendix 2: Vector Maps for Destination Vectors Suitable for shRNA Expression
Note: in all vectors shown in Appendix 2, the RFB feature contains the attR1-ccdB-attR2 elements that define an expression vector as a gateway-compatible destination vector. L11DDLUGIPXA Full sequence for this plasmid is available at the AfCS data center (http://www.signaling-Gateway.org/data/Data.html). Follow the Plasmid Database: Web interface link in the Resources section and use the above bar code to retrieve plasmid data.
XhoI
XcmI
XbaI
SnaBI
SgrAI
RsrII
PmeI
PaeR7I
PacI
NdeIMluIFspI
FseI
EcoRI
Bsu36I
BstEII
BsiWI
BaeI-bBaeI-a
AhdI
AgeI
AfeI
0.0kb1.0kb
2.0kb
3.0kb
4 .0k
b
5.0kb
6.0kb7.0kb
8.0kb
9 .0kb
10. 0
kb
11.0
kb
12.0kbCMV/LTR psi
RRE
FLA
P
RF
B
Ubi-c
IRESPuro
W
RE
SIN
/LTR
SV
4 0
ORI
GFP
Amp
pDSL_hpUGIP13066 bp
A Vector Set for Plasmid-Based RNAi Studies in Mammalian Cells AfCS Reports, Version 1.0, May 2004 Page 14
L24DDLUGIHXA Full sequence for this plasmid is available at the AfCS data center (http://www.signaling-Gateway.org/data/Data.html). Follow the Plasmid Database: Web interface link in the Resources section and use the above bar code to retrieve plasmid data.
XhoI
XcmI
XbaI
SnaBI
SgrAI
RsrIIPshAI
PmeI
PaeR7I
PacI
MluIFspI
FseI
Bsu36I
BlpI
BaeI-bBaeI-a
AhdI
AgeI
AfeI
0.0kb1.0kb
2.0kb
3 .0kb
4.0
kb
5 .0kb
6.0kb7.0kb
8.0kb
9.0kb
10 .0kb
11.0
kb
12.0k
b
13.0kb
CMV/LTR psi
RRE
FLAP
RF
B
Ubi-c
IRESHygB
WRE
SIN
/LTR
SV
40
OR I
GFP
Amp
pDSL_hpUGIH13654 bp
A Vector Set for Plasmid-Based RNAi Studies in Mammalian Cells AfCS Reports, Version 1.0, May 2004 Page 15
L20DDLHPUCXA Full sequence for this plasmid is available at the AfCS data center (http://www.signaling-Gateway.org/data/Data.html). Follow the Plasmid Database: Web interface link in the Resources section and use the above bar code to retrieve plasmid data.
XhoI
XcmI
XbaI
SnaBI
SgrAI
PspOMI
PmeI
PciI
PaeR7IPacI
NheI
NdeIMluIFspI
FseI
EcoRI
BstEII
BsmBI
BlpIBaeI-bBaeI-a
ApaI
AhdI
AfeI
0.0kb1.0kb
2.0kb
3 .0 kb
4.0k
b
5.0kb
6.0kb
7.0kb
8.0kb
9. 0
kb
1 0.0
kb
11.0k
b CMV/LTR psi
RRE
FLAP
RF
B
Ub i-c
delta C D4
WRE
SIN
/LTR
SV
40
ORI
Amp
pDSL_hpUC12384 bp
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L12DDLHPUPXA Full sequence for this plasmid is available at the AfCS data center (http://www.signaling-Gateway.org/data/Data.html). Follow the Plasmid Database: Web interface link in the Resources section and use the above bar code to retrieve plasmid data.
XhoI
XcmI
XbaI
Tth111I
SnaBI
SgrAI
RsrII
PspOMI
PmeI
PflFI
PciI
PaeR7I
PacI
NdeIMluI
FspI
FseI
EcoRI
Bsu36I
BstEII
BsiWI
BaeI-bBaeI-a
ApaI
AhdI
AgeI
AfeI
0.0kb
1.0kb
2.0kb
3.0
kb
4.0k
b
5.0kb
6.0kb
7.0kb
8.0kb
9.0
kb
10.0
kb
11.0kb
CMV/LTR psi
RREF
LAP
RF
Bcc
dB
Ubi-c
Puro
WRE
SIN/LTR
SV
40
OR
I
Amp
pDSL_hpUP11673 bp
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L06DDLHPUGXA Full sequence for this plasmid is available at the AfCS data center (http://www.signaling-Gateway.org/data/Data.html). Follow the Plasmid Database: Web interface link in the Resources section and use the above bar code to retrieve plasmid data.
XhoI
XcmI
XbaI
SnaBI
SgrAI
PspOMI
PshAI
PmeI
PciI
PaeR7IPacI
NdeIMluIFspI
FseI
EcoRI
Bsu36I
BsmBI
BaeI-bBaeI-a
ApaI
AhdI
AgeI
AfeI
0.0kb1.0kb
2.0kb
3.0 k
b
4.0k
b
5.0kb
6.0kb
7.0kb
8.0kb
9 .0 k
b
10.0
kb
11.0kb
CMV/LTR psi
RREF
L AP
RF
B
Ubi -cWRE
SIN/LTR
SV
40
ORI
GFP
Amp
pDSL_hpUG11707 bp
A Vector Set for Plasmid-Based RNAi Studies in Mammalian Cells AfCS Reports, Version 1.0, May 2004 Page 18
B03D0CXGHPXA Full sequence for this plasmid is available at the AfCS data center (http://www.signaling-Gateway.org/data/Data.html). Follow the Plasmid Database: Web interface link in the Resources section and use the above bar code to retrieve plasmid data.
XmnI
XhoIXbaI
StuI
SpeI
SnaBI
SfiI
SapI
RsrII
PvuI
PspOMI
PshAI
PflMI
PciI
PaeR7IHindIII
FspI
BtrI
BstZ17I
BstXI
BstBI
BsiWI
AseI
AscI
ApaI
AflIII
0.0kb
1.0kb
2.0 kb
3.0k
b
4.0kb
5.0kb
6 .0kb
7. 0
kb
8.0kb5' LTR PackagingSignal
Puro
CM
V
RFB
3 ' LTR
Amp
GFP
pDS_CXGhp8719 bp
A Vector Set for Plasmid-Based RNAi Studies in Mammalian Cells AfCS Reports, Version 1.0, May 2004 Page 19
B88DDSHPSCXA Full sequence for this plasmid is available at the AfCS data center (http://www.signaling-Gateway.org/data/Data.html). Follow the Plasmid Database: Web interface link in the Resources section and use the above bar code to retrieve plasmid data.
XmnI
XmaI
XhoI
SmaI
SfiI
SalI
SacI
PsiI
PciI
PaeR7I
HindIII
FspI
ClaIBtrI
Bsu36I
BstZ17I
BstEII
BssHII
BspEI
BspDI
BsgI
BglII
BclIBbvCI
AvrII
AhdI
AflIII
AflII
0.0kb0.5kb
1.0kb
1. 5 kb
2.0k
b
2.5kb
3.0kb
3 .5kb
4.0kb
4.5
k b
5.0k
b
5.5kb
del taC
D4
SV40
poly
A
Amp
RFB
SV40 prom
pDS_hpSC6026 bp
A Vector Set for Plasmid-Based RNAi Studies in Mammalian Cells AfCS Reports, Version 1.0, May 2004 Page 20
B94DDSHPCGXK Full sequence for this plasmid is ava ilable at the AfCS data center (http://www.signaling-Gateway.org/data/Data.html). Follow the Plasmid Database: Web interface link in the Resources section and use the above bar code to retrieve plasmid data.
XhoI
XcmI
XbaI
Tth111I
StuI
SnaBI
SfoI
SfiI
SexAI
ScaI
SacII
SacI
RsrII
PspOMI
PflMI
PflFI
PaeR7I
NheI
NdeI
NarI
MfeI
KpnI
KasI
HpaI
HindIII
FspI
EcoRI
EcoRI
EcoO109I
DraIII
ClaI
BtrI
BstZ17I
BstXI
BssHII
BspEI
BspDI
BsmBI
BcgI-bBcgI-a
BbvCI
AseI
ApaI
AflII
AfeI
AclI
Acc65I
0.0kb0.5kb
1.0kb
1 .5k b
2 .0k
b
2.5kb
3.0kb3.5kb
4 .0kb
4.5 kb
5 .0 k
b
5.5k
b
6.0kb
CMV
GFP
SV40prom
Kan/Neo
RFB
pDS_hpCG6446 bp
A Vector Set for Plasmid-Based RNAi Studies in Mammalian Cells AfCS Reports, Version 1.0, May 2004 Page 21
B95DDSHPEYXK Full sequence for this plasmid is available at the AfCS data center (http://www.signaling-Gateway.org/data/Data.html). Follow the Plasmid Database: Web interface link in the Resources section and use the above bar code to retrieve plasmid data.
XmnI
XbaI
Tth111I
SfiI
SexAI
ScaI
RsrII
PflMI
PflFI
NheI
KpnI
HpaI HindIII
FspI
FseI
EcoRI
EcoRI
ClaI
BtrI
BstZ17I
BstXI
BssHII
BspEI
BspDI
BsmBI
BcgI-bBcgI-a
AfeI
Acc65I
0.0kb
1.0kb
2.0
kb
3.0kb4.0kb
5 .0kb
6.0k
b RFB
EF1pr
omo
ter
YFP
SV
40prom
Kan
/Neo pDS_hpEY
7065 bp
A Vector Set for Plasmid-Based RNAi Studies in Mammalian Cells AfCS Reports, Version 1.0, May 2004 Page 22
Appendix 3: Maps for Empty Vector Control Plasmids for Use with shRNA-Expressing Destination Vectors
L10GLUGIP1XA Full sequence for this plasmid is available at the AfCS data center (http://www.signaling-Gateway.org/data/Data.html). Follow the Plasmid Database: Web interface link in the Resources section and use the above bar code to retrieve plasmid data.
XhoI
XcmI
XbaI
SspISnaBI
SgrAI
ScaI
RsrII
PstI
PmeI PinAI
Pfl23II
PaeR7I
PacI
NdeI
MroI
MluI
Kpn2I
HpaI
FspI
FseI
FauNDI
EcoO65I
Eco81I
Eco47III
Eco105IEam1105I
CspICpoI
Bsu36I
BstZ17IBstXI
BstSNI
BstEII
Bst1107I
BsrGI
BspEI
Bsp1407I
BsiWI
BsiMI
BseAI
BbvCI
BaeI-bBaeI-a
AviII
Aor51HI
AhdI
AgeI
AfeI
AccIII
Acc16I
0.0kb
1.0kb
2.0kb
3.0k
b
4.0k
b
5.0kb6.0kb
7 .0kb
8.0k b
9.0k
b
10
.0kb CMV/LTR psi
RRE
FLA
P
Ub
i -c
IRESWRE
SIN /LTR
SV
4 0
ORI
GFP
P uro
Amp
pL_UGIP11353 bp
A Vector Set for Plasmid-Based RNAi Studies in Mammalian Cells AfCS Reports, Version 1.0, May 2004 Page 23
L23GLUGIH1XA Full sequence for this plasmid is available at the AfCS data center (http://www.signaling-Gateway.org/data/Data.html). Follow the Plasmid Database: Web interface link in the Resources section and use the above bar code to retrieve plasmid data.
XhoI
XcmI
XbaI
SspISnaBI
SgrAI
RsrIIPshAI
PmeI
PaeR7I
PacI
MscI
MluI
HpaI
FspI
FseI
Bsu36I
BstZ17IBstXI
BsrGI
BsmBIBlpI
BaeI-bBaeI-a
AhdI
AgeI
AfeI
0.0kb1.0kb
2.0kb
3.0
k b
4.0k
b
5.0kb
6.0kb
7.0kb
8 .0kb
9.0k
b10
.0kb
11.0kbCMV /LTR psi
RREFLA
P
Ub i
- c
IRES
HygB
WRE
SIN/LTR
SV
40
ORI
GFP
Amp
pL_UGIH11941 bp
A Vector Set for Plasmid-Based RNAi Studies in Mammalian Cells AfCS Reports, Version 1.0, May 2004 Page 24
L19GLUC001XA Full sequence for this plasmid is available at the AfCS data center (http://www.signaling-Gateway.org/data/Data.html). Follow the Plasmid Database: Web interface link in the Resources section and use the above bar code to retrieve plasmid data.
XhoI
XcmI
XbaI
SspISnaBI
SgrAI
ScaI
PspOMI
PmeI
PciI
PaeR7IPacI
NotI
NheI
NdeI
MscI
MluI
HpaI
FspI
FseI
BstZ17I
BstEII
BsrGI
BspEI
BlpI
BbvCI
BamHI
BaeI-bBaeI-a
ApaI
AhdI
AfeI
0.0kb
1.0kb
2.0kb
3 .0
kb
4 .0kb
5.0kb6.0kb
7 .0kb
8.0kb
9.0k
b
10.0kb
CMV/LTR psi
RREF
LAP
Ubi
-c
delta CD4WRE
SIN/LTR
SV
40
OR I
Amp
pL_UC10671 bp
A Vector Set for Plasmid-Based RNAi Studies in Mammalian Cells AfCS Reports, Version 1.0, May 2004 Page 25
L17GLUP001XA Full sequence for this plasmid is available at the AfCS data center (http://www.signaling-Gateway.org/data/Data.html). Follow the Plasmid Database: Web interface link in the Resources section and use the above bar code to retrieve plasmid data.
XhoI
XcmI
XbaI
Tth111I
SspI
SnaBI
SgrAI
ScaI
RsrII
PstI
PspOMI
PmeI PflFI
PciI
PaeR7I
PacI
NotI
NdeIMluI
HpaI
FspI
FseI
Bsu36I
BstZ17I
BstXI
BstEII
BspMI
BspEIBsiWI
BbvCI
BaeI-bBaeI-a
ApaI
AhdI
AgeI
AfeI
0.0kb
1.0kb
2 .0kb
3 .0k
b
4.0kb
5.0kb
6.0kb
7 .0 kb
8.0k
b
9.0kb
CMV /LTR psi
RRE
FLA
P
Ubi
-c
PuroWRE
SIN/LTR
SV
40
OR
I
Amp
pL_UP9960 bp
A Vector Set for Plasmid-Based RNAi Studies in Mammalian Cells AfCS Reports, Version 1.0, May 2004 Page 26
L01GLUG001XA Full sequence for this plasmid is available at the AfCS data center (http://www.signaling-Gateway.org/data/Data.html). Follow the Plasmid Database: Web interface link in the Resources section and use the above bar code to retrieve plasmid data.
XhoI
XcmI
XbaI
SspI
SnaBI
SgrAI
ScaI
PstI
PspOMI
PshAI
PmeI
PciI
PaeR7I
PacI
NdeI
MscI
MluI
HpaI
FspI
FseI
Bsu36I
BstZ17I
BstXI
BsrGI
BspMI
BspEI
BbvCI
BaeI-bBaeI-a
ApaI
AhdI
AgeI
AfeI
0.0kb
1.0kb
2.0kb
3.0k
b
4.0kb
5.0kb
6.0kb
7.0kb
8.0 k
b
9.0kb CMV/LTR psi
RREF LA
P
Ub i
-c
WRESIN/LTR
SV
40
ORI
GFP
Amp
pL_UG9994 bp
A Vector Set for Plasmid-Based RNAi Studies in Mammalian Cells AfCS Reports, Version 1.0, May 2004 Page 27
M0001CXG000A Full sequence for this plasmid is available at the AfCS data center (http://www.signaling-Gateway.org/data/Data.html). Follow the Plasmid Database: Web interface link in the Resources section and use the above bar code to retrieve plasmid data.
XmnI
XhoIXbaI
VspI
StuI
SspI
SpeI
SnaBI
SfiI
RsrII
PvuI
PspOMI
PshAI
Pfl23II
PciI
NspVNotI
MroI
Kpn2I
HindIII
FspI
EcoRI
Eco147I
Eco105I
CspI
Csp45I
CpoI
ClaIBsu15I
BstSNI
Bsp120I
Bsp119I
BsiWI
BsiMIBseAI
BglII
BamHI
AviII
AsnIAseI
ApaI
AflIII
AccIII
Acc16I 0.0kb
1.0kb
2.0
kb
3.0kb4.0kb
5.0k
b
6.0k
b
5 ' LTR
PackagingS
ignal
Pur
o
CM
V
3' LTR
Amp
GFP
pCXG7004 bp