Thermo Fisher Scientific pLenti Hygro-UAS-bla Vector Owner's manual

Brand: Thermo Fisher Scientific

Model: pLenti Hygro-UAS-bla Vector

Type: Owner's manual

pLenti-hygro/UAS-bla Vector User Guide

Cat. no. K1297

K1297.pps Rev Date: 13 June 2007 Shipping Condition: Dry Ice Storage: –20°C

For Technical Support for this or any other Invitrogen Drug Discovery Solutions Products, dial 760 603 7200, extension 40266

Invitrogen Corporation • 1600 Faraday Avenue • Carlsbad, CA 92008 • Phone: 760 603 7200 • FAX: 760 602 6500 • www.invitrogen.com

TABLE OF CONTENTS

1.0 Materials Supplied ..................................................................................................................................................1

2.0 Materials Required, but Not Supplied.....................................................................................................................1

3.0 Overview.................................................................................................................................................................2

3.1 Description....................................................................................................................................................2

3.2 Map of the Vector..........................................................................................................................................3

3.3 Features of the Vector...................................................................................................................................4

4.0 Methods..................................................................................................................................................................5

4.1 Using the Vector Directly for Lentivirus Production ......................................................................................5

4.2 Propagating the Vector.................................................................................................................................5

4.3 Analyzing Transformants..............................................................................................................................5

4.4 Isolating Lentiviral Plasmid DNA...................................................................................................................6

5.0 Virapower™ Lentiviral expression system overview................................................................................................7

5.1 ViraPower™ Lentiviral Expression System....................................................................................................7

6.0 Biosafety level and handling...................................................................................................................................7

7.0 Related Products ....................................................................................................................................................8

8.0 References..............................................................................................................................................................8

9.0 Purchaser notification .............................................................................................................................................9

1.0 MATERIALS SUPPLIED

Description: pLenti-hygro/UAS-bla Vector

Shipping Condition: Dry Ice

Storage Condition: –20°C

Quantity: 10 µg in TE buffer

2.0 MATERIALS REQUIRED, BUT NOT SUPPLIED

Reagents Recommended Source Cat. no.

One Shot® Stbl3™ Chemically Competent E. coli Invitrogen C7373-03

Ampicillin Invitrogen 11593-027

293FT Cell line Invitrogen R700-07

ViraPower™ Lentiviral Packaging Mix Invitrogen K4975-00

Lipofectamine™ 2000 Transfection Reagent Invitrogen 11668-027

11668-019

Hygromycin B Invitrogen 10687-010

pLenti-zeo/CMV-GAL4 DEST vector Invitrogen K1280

Invitrogen • pLenti-hygro/UAS-bla Vector User Guide • K1297.pps Page 2 of 10

For Technical Support for this or other Invitrogen Drug Discovery Solutions Products, dial 760 603 7200, extension 40266

3.0 OVERVIEW

3.1 Description

The pLenti-hygro/UAS-bla vector contains the beta-lactamase reporter gene under the transcriptional control of

the Upstream Activator Sequence (UAS) response element. This vector can be used with the ViraPower™

Lentiviral Expression System to create a lentiviral stock. The lentiviral stock can then be used to transduce any

cell line of interest to become a UAS-bla cell line.

The Gal4-UAS system can be used for reporter gene assays. Gal4 is a commonly-used yeast transcriptional

activator, and the Gal4 DNA-binding domain (DBD) is capable of binding to UAS. Beta lactamase is a powerful

reporter gene that allows for ratiometric reporter responses with minimal experimental noise, and is expressed

when the Gal4 DBD binds to the UAS response element.

This vector is compatible with our pLenti-zeo/CMV-GAL4 DEST vector (available separately, Cat no. K1280),

which is a Gateway®-ready destination vector that contains the Gal4 DBD controlled by the human

cytomegalovirus (CMV) immediate early promoter. For example, a nuclear receptor ligand binding domain

(LBD) of interest can be fused to Gal4 DBD in the pLenti-zeo/CMV-GAL4 DEST vector. The vector is transfected

into a UAS-bla cell line, and agonist binding to the nuclear receptor LBD would result in Gal4 DBD binding to the

UAS, and expression of beta lactamase..

To use the ViraPower™ Lentiviral Expression System to create a UAS-bla cell line, simply:

1) Co-transfect the pLenti-hygro/UAS-bla vector and the ViraPower™ packaging mix into the 293FT cell

line to produce lentivirus

2) Transduce the cell line of your choice with your lentiviral stock.

Note: DO NOT transfect pLenti-hygro/UAS-bla vector into your cell line using standard lipid transfection

reagents. After transduction with lentivirus, use Hygromycin selection to obtain a stable cell line, if

desired. Refer to the ViraPower™ Lentiviral Expression System manual for more details.

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3.2 Map of the Vector

Comments for pLenti4-hygro UAS-Bla

8011 nucleotides

RSV/5 LTR hybrid promoter: bases 1- 410

RSV promoter: bases 1- 229

HIV-1 5 LTR: bases 230 - 410

HIV-1 psi (y) packaging signal: bases 521 - 565

HIV-1 Rev response element (RRE): bases 1075 - 1308

7x UAS-bla element: bases 1805 - 1972

Beta-lactamase gene: bases 2162 - 2965

SV40 early promoter: bases 3022 - 3300

EM7 promoter: bases 3349 - 3415

Hygromycin resistance gene: bases 3416 - 4441

DU3/3 LTR: bases 4531 - 4766

DU3: bases 4531 - 4585

3 LTR: bases 4586 - 4766

SV40 polyadenylation signal: bases 4837 - 4968

bla promoter: bases 5827 - 5925

Ampicillin (bla) resistance gene: bases 5926 - 6786

pUC origin: bases 6931 - 7604

pLenti4-hygro

UAS-Bla

8011 bp



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3.3 Features of the Vector

The pLenti-hygro/UAS-bla vector contains the following elements:

Feature Benefit

7X UAS Upstream Activator Sequence (UAS) response element. A gene of

interest fused to the Gal4 DNA binding domain can bind to the UAS

and drive the expression of beta-lactamase

Beta-lactamase Reporter gene that allows for ratiometric reporter responses with

minimal experimental noise

SV40 early promoter and origin Allows high-level expression of the selection marker and episomal

replication in cells expressing the SV40 large T antigen.

EM7 promoter Synthetic prokaryotic promoter for expression of the selection marker

in E. coli.

Hygromycin resistance gene Permits selection of stably transduced mammalian cell lines

∆U3/HIV-1 truncated 3’ LTR Allows viral packaging but self-inactivates the 5′ LTR for biosafety

purposes (Dull et al., 1998). The element also contains a

polyadenylation signal for transcription termination and

polyadenylation of mRNA in transduced cells.

SV40 polyadenylation signal Allows transcription termination and polyadenylation of mRNA.

bla promoter Allows expression of the ampicillin resistance gene.

Ampicillin resistance gene (β-

lactamase)

Allows selection of the plasmid in E. coli.

pUC origin Permits high-copy replication and maintenance in E. coli.

Rous Sarcoma Virus (RSV)

enhancer/promoter

Allows Tat-independent production of viral mRNA (Dull et al., 1998).

HIV-1 truncated 5′ LTR Permits viral packaging and reverse transcription of the viral mRNA

(Luciw, 1996).

5′ splice donor and 3′ acceptors Enhances the biosafety of the vector by facilitating removal of the ψ

packaging sequence and RRE such that expression of the gene of

interest in the transduced host cell is no longer Rev-dependent (Dull et

al., 1998).

HIV-1 psi (ψ) packaging signal Allows viral packaging (Luciw, 1996).

HIV-1 Rev response element (RRE) Permits Rev-dependent nuclear export of unspliced viral mRNA

(Kjems et al., 1991; Malim et al., 1989).

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4.0 METHODS

4.1 Using the Vector Directly for Lentivirus Production

You may use the pLenti-hygro/UAS-bla vector directly as supplied for transfection into 293FT cells to produce

lentivirus using the ViraPower™ Lentiviral Expression System. You may also propagate and purify vector DNA as

described below.

4.2 Propagating the Vector

To propagate and maintain the pLenti-hygro/UAS-bla vector, we recommend using One Shot® Stbl3™ Chemically

Competent E. coli (available separately from Invitrogen) to reduce the likelihood of unwanted recombination.

Select for transformants on LB agar plates containing 100 μg/ml ampicillin, and analyze the transformants as

described below.

4.3 Analyzing Transformants

We recommend analyzing the transformants using restriction digestion analysis. This ensures that aberrant

recombination has not occurred between the plasmid LTRs of the pLenti-hygro/UAS-bla vector. You will screen

colonies by performing miniprep DNA isolation and restriction analysis to validate the clones. After verifying the

correct clones, you will use the miniprep DNA to re-transform E. coli. You will then isolate plasmid DNA for

transfection and lentivirus production. Plasmid DNA for transfection into 293FT cells must be very clean and free

from contaminants and salts, and should be isolated by midiprep.

Important: We recommend using E. coli that is wild type for endonuclease 1 (endA1+). When performing

plasmid DNA isolation with commercially available kits, ensure that Solution I of the Lysis buffer (often

called Resuspension Buffer) contains 10 mM EDTA to inactivate the endonuclease to avoid DNA nicking

and vector degradation. Alternatively, follow the instructions included in the plasmid purification kits for

endA1+ E. coli strains. We recommend using the PureLink™ HQ Mini Plasmid Purification Kit and

preparing lentiviral plasmid DNA using S.N.A.P.™ MidiPrep Kits.

Restriction Digest Guidelines

To confirm that no rearrangement in the LTR regions has taken place, perform restriction digests using a

combination of Afl II and Cla I. Afl II sites are present in both LTRs, and the Cla I site is present before the 7X UAS

sequence. After digestion with these enzymes, 3 DNA fragments are generated from the Afl II + Cla I digest. Any

unexpected DNA fragments are a result of LTR recombination.

Materials Needed

You will need the following materials:

• LB medium containing 100 µg/ml ampicillin

• PureLink™ HQ Mini Plasmid Purification Kit or equivalent

• Afl II and Cla I restriction enzymes and buffers

• E-Gels® 1.2% agarose gels or equivalent

• S.N.A.P.™ MidiPrep Kit or equivalent

Screening Colonies by Miniprep

For each transformation, pick 10-20 colonies from plates obtained after plating the transformation mix. Culture

them overnight in LB medium containing 100 µg/ml ampicillin.

Isolate plasmid DNA using PureLink™ HQ Mini Plasmid Purification Kit or equivalent. The typical yield of

pLenti DNA with PureLink™ HQ Mini Plasmid Purification Kit is ~ 5-7 µg, which is lower than the average DNA

yield using this purification kit. Perform restriction digests on plasmid DNA, and analyze the digested DNA on

0.8% or 1.2% agarose gels to confirm the correct clones.

What You Should See

Depending on the restriction sites you are using, you should be able to determine the number and size of bands

you should obtain from your digestion. Agarose gel analysis should show the correct digestion pattern.

Additional or unexpected bands indicate aberrant recombination of the lentiviral vector.

Invitrogen Corporation • 1600 Faraday Avenue • Carlsbad, CA 92008 • Phone: 760 603 7200 • FAX: 760 602 6500 • www.invitrogen.com

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4.4 Isolating Lentiviral Plasmid DNA

This protocol provides general steps to retransform E. coli and perform isolation of plasmid DNA for lentivirus

production. pLenti plasmid DNA midipreps often have lower yields; therefore, a 100 ml volume of culture must

be used for one DNA midiprep.

1. Dilute 1 µl of miniprep plasmid DNA from a positive clone 1:500 in TE.

2. Use 1 µl of this diluted DNA to retransform competent E. coli.

3. Plate approximately one-tenth of the transformation on LB plates containing 100 µg/ml ampicillin

and incubate at 37°C overnight.

4. Pick 1 colony and culture in 2-3 ml LB medium containing 100 µg/ml ampicillin for 6-8 hours at 37°C

to obtain a starter culture.

5. Inoculate the entire volume of the starter culture into LB medium containing 100 µg/ml ampicillin

and culture at 37°C overnight.

Note: Use a 100 ml volume for large scale or midiprep isolation of DNA.

6. Isolate plasmid DNA using S.N.A.P.™ MidiPrep Kit or equivalent.

Note: For best results using the S.N.A.P.™ MidiPrep Kit, split the 100 ml culture into two 50-ml tubes

and process as if they were separate samples. Run both samples over the same DNA binding

column B, (i.e. perform two spins) and treat as a single DNA prep in subsequent steps.

7. Perform restriction analysis as described on the previous page.

8. Use the purified plasmid DNA from the positive clone for producing the lentivirus.

Note: Typical DNA yield should be ~300-400 µg and the O.D. 260/280 ratio should be between 1.8 and

2.1.

DNA Isolation Guidelines

Once you have generated and validated your clone you will isolate plasmid DNA for transfection. Plasmid DNA

for transfection into eukaryotic cells must be very clean and free from contamination with phenol and sodium

chloride. Contaminants will kill the cells, and salt will interfere with lipid complexing, decreasing transfection

efficiency. We recommend isolating lentiviral plasmid DNA using the S.N.A.P.™ MidiPrep Kit.

Important: Do not use mini-prep plasmid DNA for lentivirus production.

Maintaining the Expression Clone

Once you have generated your expression clone, maintain and propagate the plasmid in LB medium containing

100 μg/ml ampicillin.

Long Term Storage

Once you have identified the correct clone, be sure to purify the colony and make a glycerol stock for long term

storage. We recommend that you store a stock of plasmid DNA at -20°C.

1. Streak the original colony out for single colonies on LB plates containing 100 µg/ml ampicillin.

2. Isolate a single colony and inoculate into 1-2 ml of LB containing 100 µg/ml ampicillin.

3. Grow until culture reaches stationary phase.

4. Mix 0.85 ml of culture with 0.15 ml of sterile glycerol and transfer to a cryovial. Store at -80°C.

Invitrogen Corporation • 1600 Faraday Avenue • Carlsbad, CA 92008 • Phone: 760 603 7200 • FAX: 760 602 6500 • www.invitrogen.com

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5.0 VIRAPOWER™ LENTIVIRAL EXPRESSION SYSTEM OVERVIEW

5.1 ViraPower™ Lentiviral Expression System

The pLenti-hygro/UAS-bla vector may be used in combination with the ViraPower™ Lentiviral Expression System

for viral transduction of mammalian cells. The ViraPower™ Lentiviral Expression System facilitates highly

efficient delivery of genes to dividing and non-dividing mammalian cells using a replication-incompetent

lentivirus. Based on the lentikat™ system developed by Cell Genesys (Dull et. al., 1998), the ViraPower™ Lentiviral

Expression System possesses features which enhance its biosafety while allowing high-level gene expression in a

wider range of cell types than traditional retroviral systems.

The ViraPower™ Packaging Mix provides a pre-optimized mix of plasmids that produce the lentiviral packaging

proteins (gag/pol and rev) and the VSV-G envelope protein. The VSV-G envelope gives the viral particles the

ability to transduce a broad range of mammalian cells. The human 293FT Cell Line is supplied with the

ViraPower™ Lentiviral Expression kits to facilitate optimal lentivirus production (Naldini et al., 1996). The 293FT

Cell Line, a derivative of the 293F Cell Line stably and constitutively expresses the SV40 large T antigen from

pSPORTR6Tag.neo, and must be maintained in medium containing Geneticin®.

To use the ViraPower™ Lentiviral Expression System, co-transfect the pLenti-hygro/UAS-bla vector along with the

optimized ViraPower™ Lentiviral Packaging Mix into 293FT producer cells. In 24-48 hours harvest the

supernatant, which now has replication-incompetent viral particles. Since the packaging proteins are provided in

trans, these plasmids are not part of the final viral particles. You can use this supernatant to efficiently transduce

any mammalian cell. Each transfection produces sufficient supernatant for multiple transduction experiments.

ViraPowerTM 293FTTM Producer Cell Line

pLenti4-hygro

UAS-Bla

8011 bp



Harvest viral supernatant

and determine the titer

Store up to one year

Transduce mammalian cells,

select for stable expression

Cotransfect the ViraPowerTM 293FTTM

Producer Cell Line with pLenti4-hygro UAS-Bla

and the optimized ViraPowerTM packaging mix.

For more information about the ViraPower™ Lentiviral Expression System, the ViraPower™ Packaging Mix, and

the biosafety features of the system, refer to the ViraPower™ Lentiviral Expression System manual. For more

information about the 293FT Cell Line, refer to the 293FT manual. Both manuals are available for downloading

from www.invitrogen.com, or by contacting Technical Support.

6.0 BIOSAFETY LEVEL AND HANDLING

The pLenti-hygro/UAS-bla vector should be handled at Biosafety Level 1 and virus produced by this method

should be handled at Biosafety Level 2.

Handle all lentiviruses in compliance with established institutional guidelines. Since safety requirements for use

and handling of lentiviruses may vary at individual institutions, we recommend consulting the health and safety

guidelines and/or officers at your institution prior to use of the ViraPower™ Lentiviral Expression System.

The ViraPower™ Lentiviral Expression System is a third-generation system based on lentiviral vectors developed

by Dull et al., 1998 . This third-generation lentiviral system includes a significant number of safety features

designed to enhance its biosafety and to minimize its relation to the wild-type, human HIV-1 virus

For a full description of the biosafety features of the ViraPower™ Lentiviral Expression System, refer to the

product manual, available at www.invitrogen.com.

Invitrogen Corporation • 1600 Faraday Avenue • Carlsbad, CA 92008 • Phone: 760 603 7200 • FAX: 760 602 6500 • www.invitrogen.com

Invitrogen • pLenti-hygro/UAS-bla Vector User Guide • K1297.pps Page 8 of 10

For Technical Support for this or other Invitrogen Drug Discovery Solutions Products, dial 760 603 7200, extension 40266

7.0 RELATED PRODUCTS

Reagents Recommended Source Cat. no.

LB Media Invitrogen 10855-021

S.N.A.P™. MidiPrep Kit Invitrogen K1910-01

PureLink™ HQ Plasmid Miniprep Kit Invitrogen K2100-01

E-Gel® 1.2% Starter Pak (6 gels + Powerbase™) Invitrogen G6000-01

E-Gel® 1.2% 18 Pak Invitrogen G5018-01

LiveBLAzer™-FRET B/G loading kit Invitrogen K1095

ToxBLAzer™ DualScreen kit Invitrogen K1138

LyticBLAzer™-FRET B/G Homogeneous kit Invitrogen K1148

8.0 REFERENCES

Dull, T., Zufferey, R., Kelly, M., Mandel, R. J., Nguyen, M., Trono, D., and Naldini, L. (1998) A Third-Generation

Lentivirus Vector with a Conditional Packaging System. J. Virol. 72, 8463-8471.

Luciw, P. A. (1996) in Fields Virology (Fields, B. N., Knipe, D. M., Howley, P. M., Chanock, R. M., Melnick, J. L., Monath, T.

P., Roizman, B., and Straus, S. E., eds), 3rd Ed., pp. 1881-1975, Lippincott-Raven Publishers, Philadelphia, PA.

Kjems, J., Brown, M., Chang, D. D., and Sharp, P. A. (1991) Structural Analysis of the Interaction Between the Human

Immunodeficiency Virus Rev Protein and the Rev Response Element. Proc. Natl. Acad. Sci. USA 88, 683-687.

Naldini, L., Blomer, U., Gage, F. H., Trono, D., and Verma, I. M. (1996) Efficient Transfer, Integration, and Sustained Long-

Term Expression of the Transgene in Adult Rat Brains Injected with a Lentiviral Vector. Proc. Natl. Acad. Sci.

USA 93, 11382-11388.

Malim, M. H., Hauber, J., Le, S. Y., Maizel, J. V., and Cullen, B. R. (1989) The HIV-1 Rev Trans-activator Acts Through a

Structured Target Sequence to Activate Nuclear Export of Unspliced Viral mRNA. Nature 338, 254-257.

Invitrogen Corporation • 1600 Faraday Avenue • Carlsbad, CA 92008 • Phone: 760 603 7200 • FAX: 760 602 6500 • www.invitrogen.com

Invitrogen • pLenti-hygro/UAS-bla Vector User Guide • K1297.pps Page 9 of 10

For Technical Support for this or other Invitrogen Drug Discovery Solutions Products, dial 760 603 7200, extension 40266

9.0 PURCHASER NOTIFICATION

Limited Use Label License No: 5 Invitrogen Technology

The purchase of this product conveys to the buyer the non-transferable right to use the purchased amount of the product and

components of the product in research conducted by the buyer (whether the buyer is an academic or for-profit entity). The

buyer cannot sell or otherwise transfer (a) this product (b) its components or (c) materials made using this product or its

components to a third party or otherwise use this product or its components or materials made using this product or its

components for Commercial Purposes. The buyer may transfer information or materials made through the use of this product

to a scientific collaborator, provided that such transfer is not for any Commercial Purpose, and that such collaborator agrees in

writing (a) not to transfer such materials to any third party, and (b) to use such transferred materials and/or information

solely for research and not for Commercial Purposes. Commercial Purposes means any activity by a party for consideration

and may include, but is not limited to: (1) use of the product or its components in manufacturing; (2) use of the product or its

components to provide a service, information, or data; (3) use of the product or its components for therapeutic, diagnostic or

prophylactic purposes; or (4) resale of the product or its components, whether or not such product or its components are

resold for use in research. Invitrogen Corporation will not assert a claim against the buyer of infringement of patents owned or

controlled by Invitrogen Corporation which cover this product based upon the manufacture, use or sale of a therapeutic,

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components was employed, provided that neither this product nor any of its components was used in the manufacture of such

product. If the purchaser is not willing to accept the limitations of this limited use statement, Invitrogen is willing to accept

return of the product with a full refund. For information on purchasing a license to this product for purposes other than

research, contact Licensing Department, Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad, California 92008. Phone

(760) 603-7200. Fax (760) 602-6500. Email: [email protected]

Limited Use Label License No: 108 Lentiviral Technology

The Lentiviral Technology (based upon the lentikat™ system) is licensed from Cell Genesys, Inc., under U.S. Patent Nos. 5,686,279;

5,834,256; 5,858,740; 5,994,136; 6,013,516; 6,051,427; 6,165,782 and 6,218,187 and corresponding patents and applications in other

countries for internal research purposes only. Use of this technology for gene therapy applications or bioprocessing other than for

non-human research use requires a license from Cell Genesys (Cell Genesys, Inc. 342 Lakeside Drive, Foster City, California 94404).

The purchase of this product conveys to the buyer the non-transferable right to use the purchased amount of the product and

components of the product in research conducted by the buyer, including non-gene therapy research and target validation

applications in laboratory animals (whether the buyer is an academic or for-profit entity). The buyer cannot sell or otherwise transfer

(a) this product (b) its components or (c) materials made using this product or its components to a third party or otherwise use this

product or its components or materials made using this product or its components for Commercial Purposes. The buyer may transfer

information or materials made through the use of this product to a scientific collaborator, provided that such transfer is not for any

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such transferred materials and/or information solely for research and not for Commercial Purposes. Commercial Purposes means

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the limitations of this limited use statement, Invitrogen is willing to accept return of the product with a full refund. For information

on purchasing a license to this product for purposes other than research, contact Licensing Department, Invitrogen Corporation, 1600

Faraday Avenue, Carlsbad, California 92008. Phone (760) 603-7200. Fax (760) 602-6500.

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Limited Use Label License No. 150: GeneBLAzer® Technology

This product and/or its use is the subject of one or more of U.S. Patent Nos. 5,741,657, 5,955,604, 6,291,162, and 6,472,205 and foreign

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buyer cannot sell or otherwise transfer (a) this product (b) its components or (c) materials made using this product or its components

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its components, whether or not such product or its components are resold for use in research. Invitrogen Corporation will not assert a

claim against the buyer of infringement of the above patents based upon the manufacture, use, or sale of a therapeutic, clinical

diagnostic, vaccine, or prophylactic product developed in research by the buyer in which this product or its components was

employed, provided that neither this product nor any of its components was used in the manufacture of such product. If the

purchaser is not willing to accept the limitations of this limited use statement, Invitrogen is willing to accept return of the product

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Department, Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad, California 92008. Phone (760) 603-7200. Fax (760) 602-6500.

Invitrogen Corporation • 1600 Faraday Avenue • Carlsbad, CA 92008 • Phone: 760 603 7200 • FAX: 760 602 6500 • www.invitrogen.com

Thermo Fisher Scientific pLenti Hygro-UAS-bla Vector Owner's manual

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Model: pLenti Hygro-UAS-bla Vector

Type: Owner's manual

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Thermo Fisher Scientific pLenti Hygro-UAS-bla Vector Owner's manual

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