capture-tec -3 system

Capture-Tecª

pHookª-3 SystemVersion D

18111325-0048

Capture-Tecª pHookª-3 Systemfor the Isolation of Transfected Eukaryotic Cells

Catalog no. K690-01; V690-20

U.S. Headquarters: European Headquarters:Invitrogen Corporation Invitrogen BV1600 Faraday Avenue PO Box 2312, 9704 CH GroningenCarlsbad, CA 92008 The NetherlandsToll Free Tel: (800) 955-6288 Toll Free Tel: 00800 5345 5345Tel: (760) 603-7200 Toll Free Fax: 00800 7890 7890Fax: (760) 603-7201 Tel: +31 (0) 50 5299 299E-mail: [email protected] Fax: +31 (0) 50 5299 281Web site: www.invitrogen.com E-mail: [email protected]

iii

Important Information

Introduction This manual is supplied with the following products:

¥ Capture-Tecª pHookª-3 Kit, Catalog no. K690-01¥ Capture-Tecª pHookª-3 Vector, Catalog no. V690-20

Kit Contents The following table describes the components of the Capture-Tecª pHookª-3 Kit.

Component Amount

pHookª-3 plasmid 20Êµg, lyophilized

pHookª-3lacZ 10Êµg, lyophilized

Capture-Tecª Beads, 20 reactions

[4Êµm magnetic beads coated with thehapten 4-ethoxymethylene-2-phenyl-2-oxazolin-5-one (phOx)]

2 tubes, 10 reactions/tube

[Each tube contains 2.0 x 107 beads in100Êµl PBS, 0.1% bovine serum albumin,and 0.02% sodium azide]

Zeocinª, 100 mg/ml 1.25 ml (in distilled, deionized autoclavedwater)

Zeocinª is a trademark of Cayla.

Shipping and Storage

The Capture-Tecª pHookª-3 Kit is shipped at +4¡C. Upon receipt of the kit, store theplasmids and Zeocinª at -20¡C and the magnetic beads (2 tubes) at +4¡C.

Purchase ofZeocinª

125Êmg of Zeocinª has been provided in the Capture-Tecª pHookª-3 Kit, allowing you toimmediately begin your experiments. Additional Zeocinª can be purchased from Invitrogen(see next page). For your convenience, the drug is prepared in autoclaved, deionized waterand aliquoted into 1.25Êml aliquots at 100 mg/ml. The stability of Zeocinª is guaranteed forsix months, when stored at -20¡C.

Capture-Tecª

pHookª-3 VectorpHookª-3 plasmid, 20Êµg, lyophilizedpHookª-3lacZ, 10Êµg, lyophilized

Plasmids are shipped at room temperature. Upon receipt, please store at -20¡C.

continued on next page

iv

Important Information, continued

OrderingInformation

The following materials are available separately from Invitrogen. Please call ourTechnical Services Department for more information (see below).

Product Amount Catalog no.

Capture-Tecª Beads, 40 reactions 4 tubes, 10 reactions/tube

[Each tube contains 2.0 x 107 beadsin 100Êµl PBS, 0.1% bovine serumalbumin, and 0.02% sodium azide]

R665-01

Capture-Tecª Stand 1 magnetic stand R670-01

Zeocinª antibiotic 1 gram R250-01

5 gram R250-05

Anti-myc Antibody 50Êµl R950-25

HandlingZeocinª

¥ High salt and acidity or basicity inactivate Zeocinª. Reduce the salt in bacterialmedium and adjust the pH to 7.5 to keep the drug active (see Low Salt LB Medium,page 18).

¥ Store Zeocinª at -20¡C and thaw on ice before use.

¥ Zeocinª is light sensitive. Store the drug, and plates or medium containing drug, in thedark.

¥ Wear gloves, a laboratory coat, and safety glasses or goggles when handling Zeocinª-containing solutions.

¥ Do not ingest or inhale solutions containing the drug.

¥ Be sure to bandage any cuts on your fingers to avoid exposure to the drug.

Other MaterialsNeeded by User

You will need the following solutions and equipment:

¥ Strong magnet or magnetic stand (Invitrogen, Catalog no. R670-01)

¥ Tissue-culture flasks and plates

¥ Complete medium for your cell line

¥ PBS/ 3ÊmM EDTA (See Recipes, page 19)

¥ X-gal Reagent (See Recipes, page 19)

¥ Hemacytometer

¥ Rotating tube mixer

Technical Service For Technical Service, please call, write, fax or E-mail:


v

Purchaser Notification

Patent Information This product is designed and intended for research use only, not for diagnostic ormedical use.

The Molecular Hookª technology encompassing the Capture-Tecª pHookª-3 System isowned by Invitrogen Corporation, patents pending.

The technology covering the construction and use of single chain Fv recombinant antigenbinding molecules and genes thereof (collectively, sFv) is covered by U. S. Patent No.5,260,203 and other domestic and foreign patent rights, owned by Enzon, Inc. andlicensed by Invitrogen Corporation.

Limits of License The purchase of this product includes a fully paid-up, limited license under such patentrights to use this product for research use only. No license is granted for any other uses,such as (without limitation)

(1) preclinical toxicological studies with sFv or any research or commercial activitiesbeyond such toxicological studies;

(2) diagnosis or detection of disease or other health related conditions in humans, animalor plants using sFvs;

(3) therapeutic applications of any type using sFvs.

No other license is granted expressly, impliedly, or by estoppel.

For MoreInformation

For more information concerning the availability of additional licenses for other thanresearch use, contact:

¥ Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad, CA 92008 for MolecularHookª technologies.

¥ Enzon, Inc., 20 Kingsbridge Road, Piscataway, New Jersey 08854 for single chainantigen binding molecules.

vi

Material Safety and Data Sheet (MSDS)

Introduction In an effort to provide our customers with the best possible information any time theyneed it, MSDSs are now available 24 hours a day, 7 days a week from either our website or the 3E Company (see below).

MSDSInformationOn-line

Chemicals that require MSDS information are listed on our web site at the followingaddress:

http://www.invitrogen.com/tech/index.html

Just click on ÒChoose a Tech Service PageÓ and select ÒSafety & MSDSÓ. MSDSinformation is available by compound name, product catalog number, or compound partnumber listed in the upper right hand corner of the label.

MSDSInformation byPhone

Customers of Invitrogen can call the 3E Company, 24 hours a day, 7 days a week for MSDSinformation. This information can be obtained directly over the phone, faxed, or mailed tothe customer. MSDS information can be accessed by compound, product catalog number, orcompound part number listed in the upper right hand corner of the label.

3E Company4920 Carroll Canyon RoadSan Diego, CA 92121Voice: 1-800-451-8346 (U.S., Canada, and Guam)Voice: 1-619-677-0150 (all other countries)Fax: 1-619-677-0270(See below for other toll-free numbers)

EmergencyInformation

In the event of an emergency, the 3E Company can help with disposal or spillinformation. They can also connect the customer with poison control or the Universityof California at San Diego Medical Center doctors.

Toll-FreeNumbers

If you reside in one of the countries listed below, you can contact the 3E Companyusing the toll-free number listed.

Country Toll-Free Number Country Toll-Free Number

Belgium 008071178 Mexico 958008342735

China 108001100017 958004518346

France 0800903046 958003603220

Germany 0130829154 958003466737

Indonesia 00180316570620 Netherlands 08000220091

Italy 167870990 Singapore 8001100987

Japan 006633800185 Spain 900931263

Korea 0308116570820 Turkey 00800136570900

Malaysia 1800808714 United Kingdom 0800967491

vii

Table of Contents

Important Information................................................................................................................ iii

Purchaser Notification...................................................................................................................v

Material Safety and Data Sheet (MSDS) ....................................................................................vi

Table of Contents ........................................................................................................................ vii

Introduction....................................................................................................................................1

Overview .........................................................................................................................................................................1pHookª-3 ........................................................................................................................................................................4

Methods...........................................................................................................................................6

FastStart ..........................................................................................................................................................................6Cloning into pHookª-3 ...................................................................................................................................................7Transformation into E. coli .............................................................................................................................................9Transfection and Cell Selection ....................................................................................................................................11Optimization of Transfection and Selection..................................................................................................................15Technical Assistance .....................................................................................................................................................17

Appendix.......................................................................................................................................18

Recipes ..........................................................................................................................................................................18Zeocinª .........................................................................................................................................................................20pHookª-3lacZ ...............................................................................................................................................................22Technical Service ..........................................................................................................................................................23References .....................................................................................................................................................................25

1

Introduction

Overview

Introduction The Capture-Tecª pHookª-3 System allows rapid selection and isolation of transientlytransfected cells from total populations of transfected and untransfected cells. Thissystem utilizes a specially designed vector (pHookª-3) that uses the Rous sarcoma virus(RSV) promoter to express and display a single-chain antibody (sFv) against a specifichapten on the surface of transfected cells. Cells expressing the sFv can then be isolatedfrom the culture by binding to hapten-coated magnetic beads (Chesnut et al., 1996). Thismeans you can use the Capture-Tecª pHookª-3 System to study:

¥ The effect of dominant, negative mutations of growth regulating proteins or toxic geneproducts

¥ The acute effects of constitutive mutations

¥ The effect of anti-sense RNA or ribozymes

¥ Temporal events along a developmental or signaling pathway

¥ Activation or inactivation of downstream genes using differential display

You may also use the Capture-Tecª pHookª-3 System to isolate a homogeneouspopulation of transfected cells for:

¥ Immunoblot analysis of proteins

¥ Isolation of DNA or RNA

¥ Flow Cytometry

Flow cytometry of propidium iodide-stained nuclei has been performed on lysed,selected cells without removal of the beads using the following equipment:

Beckton Dickinson FACS Sort, Epic 752, and Coulter XL.

Check with the manufacturer of your flow cytometry equipment for compatibility withthe 4Êµm magnetic beads.

Brief Descriptionof the Capture-Tecª pHookª-3System

pHookª-3 encodes a single-chain antibody (sFv) directed toward the hapten 4-ethoxy-methylene-2-phenyl-2-oxazolin-5-one (phOx) (Griffiths et al., 1984; Hoogenboom et al.,1991). The signal peptide from the murine Ig k-chain V-J2-C region is fused in front ofcoding region of the sFv to direct the antibody to the plasma (Coloma et al., 1992; Kabatet al., 1987). The antibody is fused at the C-terminus to the transmembrane domain fromthe platelet derived growth factor receptor (PDGFR), allowing the antibody to be anchoredand displayed on the extracellular side of the plasma membrane (Gronwald et al., 1988).Transfected cells expressing the sFv can be isolated from whole cultures by usingmagnetic beads coated with phOx and a strong magnet or magnetic stand (see page iv).When your gene of interest is inserted into the multiple cloning site of pHookª-3,transiently transfected populations of cells expressing the gene of interest can be isolatedand studied for acute and chronic changes in cellular physiology even if the expressedgene inhibits cell growth.


2

Overview, continued

Flow Chart ofTransfection,Expression, andIsolation

pHookª-36.2 kb

Co

lE1

BGH pA

SV

40

Signal Peptide

PDGFR Transmem-brane DomainphOx sFvH

A

myc

mycPCMV PRSVTKpAGene of Interest

Zeocin EM-7

earlySV40 pA

MagneticBead

PhOx

Select cells expressing your gene and the single-chain antibody using a magnet.

4.

Express your gene and the single-chain antibody. sFv is secreted and displayed on the surface of the cell.

2.

TM

TM

Plasmamembrane

Add magnetic beads coated with phOx and allow cells to bind.

3.

Transmembranedomain

Clone your gene of interest into pHookª-3 vector. Transfect into cells.1.

pHookª-36.2 kb

Col

E1BGH pA

SV

40

Signal Peptide


A

myc

mycPCMV PRSVTKpAGene of Interest

Zeocin EM-7

earlySV40 pA

f1 ori

f1 ori


3

Overview, continued

Mechanism ofAction of Zeocinª

pHookª-3 contains a resistance marker (Sh ble) to the antibiotic Zeocinª. Zeocinª is amember of the bleomycin family of cytotoxins that kills a wide range of prokaryotic andeukaryotic cells by binding chromosomal DNA and causing random double-strandedbreaks. The Zeocinª resistance gene, ble, has been isolated from Streptoalloteichushindustanus. The Sh ble gene product inactivates Zeocinª by binding directly to it in astoichiometric fashion and preventing chromosomal DNA from being bound and cleaved.The Zeocinª resistance gene is included in this cloning vector as an alternative to b-lac-tamase. Since the Zeocinª resistance protein is overproduced in the cytoplasm and notsecreted like b-lactamase, there is no danger of ZeoS satellite colonies arising. For moreinformation on Zeocinª, please see page 20.

4

pHook™-3

Description pHookª-3 is a 6.2 kb plasmid encoding a single-chain antibody fusion directed to thehapten phOx (4-ethoxymethylene-2-phenyl-2-oxazolin-5-one). This antibody fusion isexpressed from the RSV promoter and displayed on the extracellular side of the plasmamembrane. Genes cloned into pHookª-3 are expressed from the CMV promoter for high-level expression.

Features andBenefits ofpHookª-3

The table below describes the features and benefits of the pHookª-3 plasmid. Allfeatures have been functionally tested.

Feature Benefit

phOx sFv This single-chain antibody recognizes thehapten, phOx, and allows isolation or detectionof cells displaying this sFv (Griffiths et al.,1984; Hoogenboom et al., 1991)

Rous sarcoma virus (RSV) promoter Permits high-level expression of the sFv in awide variety of eukaryotic cells (Gorman et al.,1982)

Signal peptide

(Met-Glu-Thr-Asp-Thr-Leu-Leu-Leu-Trp-Val-Leu-Leu-Leu-Trp-Val-Pro-Gly-Ser-Thr-Gly-Asp)

Signal peptide from murine Ig k-chain V-J2-Cregion directs the sFv to the plasma membranefor extracellular display (Coloma, et al., 1992;Kabat, et al., 1987)

Hemagglutinin A epitope tag

(Tyr-Pro-Tyr-Asp-Val-Pro-Asp-Tyr-Ala)

Allows detection of the sFv by monoclonalantibody 12CA5 (Kolodziej and Young, 1991;Niman et al., 1983)

myc epitope tag

(Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu)

Allows detection of the sFv by the Anti-mycAntibody to the c-myc epitope (Catalog no.R950-25) (Evans et al., 1985)

Platelet-derived growth factorreceptor transmembrane domain(PDGFR-TM)

Fusion of PDGFR-TM to sFv anchors theantibody to the plasma membrane for display

Bovine growth hormone (BGH)polyadenylation signal

Permits proper processing and polyadenylationof the mRNA for stabilization of the message(Goodwin et al., 1992)

Cytomegalovirus (CMV) immediateearly promoter

Permits high-level expression of the gene ofinterest (Boshart et al., 1985)

Multiple cloning site (MCS) Permits insertion of gene of interest

Thymidylate kinase (TK)polyadenylation site

Permits proper processing and polyadenylationof the mRNA for stabilization of the message

Zeocinª resistance gene Allows selection of the plasmid in E. coli

Note: this gene will also confer resistance toZeocinª in mammalian cells

pMB1 (pUC-derived) origin High copy replication and growth in E.Êcoli


5

pHook™-3, continued

Map of pHookª-3 The figure below summarizes the features of the pHookª-3 vector. The completesequence of the vector is available for downloading from our World Wide Web site(htpp://www.invitrogen.com) or by contacting Technical Service (see page 23).

Comments for pHook™-3: 6199 nucleotides

SV40 origin and promoter: bases 6-785EM-7 promoter: bases 786-834Sh ble gene (ZeoR): bases 854-1228Early SV40 polyadenylation signal: bases 1292-1421pMB1 (pUC-derived) origin: bases 1531-2204CMV promoter: bases 2263-2881pHook™-3 Forward priming site: bases: 2866-2884Multiple cloning site: bases 2931-3001pHook™ Reverse priming site: bases: 3040-3058TK polyadenylation signal: bases 3198-3566RSV LTR: bases 4003-4355Murine Ig secretion signal: bases 4366-4428Hemagglutinin A epitope: bases 4429-4455phOx sFv: bases 4471-5184myc epitope 1: bases 5194-5226myc epitope 2: bases 5239-5271PDGFR transmembrane domain: bases 5272-5421BGH polyadenylation signal: bases 5483-5711f1 origin: bases 5774-6187

SignalPeptide


A

myc

mycPCMV PRSVTKpA

pMB1 ori

BGH pA

SV

40

Hin

d III

Asp

718

IK

pn I

Bgl

IID

ra I

Nhe

IX

ba I

Bst

X I

Zeocin EM-7

f1oripHook ™-3

6.2 kb

SV40

pA

6

Methods

FastStart

Introduction The instructions below will allow you to quickly get started transfecting and selectingyour cells once you have cloned your insert into the pHookª-3 vector (see page 7 for adetailed map of the multiple cloning site).

¥ For selection of transformants on Zeocinª, see page 9.

¥ For recommendations on plasmid preparation, see page 11.

¥ For more information about transfection and selection, see pages 11-14.

¥ To optimize transfection and selection using the control plasmid pHookª-3lacZ, seepages 15-16.

Transfection ofCells

Be sure to have a method for transfecting your cell line before starting.

1. Transfect 2 x 106 cells with 1-5 µg of your pHookª-3 construct.

2. Plate out your cells in 60Êmm plates and incubate 2-48Êhours posttransfection.

Selection of Cells In general, we recommend using PBS/3ÊmM EDTA (see Recipes, page 19) to harvestyour cells; however trypsin/EDTA may be tried at a trypsin concentration of 0.05%. Beaware that trypsin may digest the sFv causing decreased selection efficiency.

1. When you are ready to select cells, vortex the magnetic bead slurry and aliquot10Êµl (2.0 x 106 beads) to a 1.5Êml microcentrifuge tube. Use one tube for each60Êmm plate of cells.

2. Wash beads with 1Êml complete medium. Pellet beads with a magnet or magneticstand and remove supernatant.

3. Take a 60Êmm plate containing transfected cells and harvest the cells usingPBS/3ÊmM EDTA. Pellet the cells and decant the supernatant. Resuspend cells in1Êml complete medium (containing serum and any other necessary supplements).

4. Add the 1Êml cell suspension to a tube containing Capture-Tecª beads. Incubate30-60 minutes at 37¡C on a slow rotator (5-10 revolutions per minute).

5. To select cells, place the tubes containing the bead-cell mixture in a magnetic standand mix for 30 seconds to 1 minute with gentle end-over-end rotation.

6. Remove the non-selected cells with a pipet.

7. Remove the tubes from the magnetic stand and resuspend the beads and cells in1Êml complete medium. Vortex gently to resuspend cells.

8. Pellet beads and bound cells using the magnetic stand and pipet off the supernatant.

9. Repeat Steps 7 and 8 two more times.

10. Resuspend selected cells in 100Êµl complete medium and count your cells. Cells areready to culture or analyze.

ImportantThe magnetic beads cannot be removed efficiently from the cells. The presence of thebeads does not appear to interfere with cell growth after selection (see page 14).

7

Cloning into pHook™-3

Cloning intopHookª-3

The graphic below shows the RSV promoter and the multiple cloning site to help youclone into pHookª-3.

GGAGACCCAA GCTTGGTACC AGATCTTTTA AAGCTAGCTC TAGAGTACCG AGCTCGGATC

ACGCCCCAGC ACTCGTCCGA GGGCAAAGGA ATAGGGGAGA TGGGGGAGGC TAACTGAAAC

ACGGTGGGAG GTCTATATAA GCAGAGCTCG TTTAGTGAAC CGTCAGATCG CCTGGAGACG

pHookª Reverse priming site

Putative transcriptional start

Hind III

5« end of hCMV promoter/enhancer

enhancer region (5« end)

AP1

CAAT

TATA

3« end of hCMV

enhancer region (3« end)

pHookª-3 Forward priming site

Asp718 I Kpn I Bgl II Dra I

BstX I

HSV TK poly (A) addition site

CGTTACATAA CTTACGGTAA ATGGCCCGCC TGGCTGACCG CCCAACGACC CCCGCCCATT

GACGTCAATA ATGACGTATG TTCCCATAGT AACGCCAATA GGGACTTTCC ATTGACGTCA

ATGGGTGGAG TATTTACGGT AAACTGCCCA CTTGGCAGTA CATCAAGTGT ATCATATGCC

AAGTACGCCC CCTATTGACG TCAATGACGG TAAATGGCCC GCCTGGCATT ATGCCCAGTA

CATGACCTTA TGGGACTTTC CTACTTGGCA GTACATCTAC GTATTAGTCA TCGCTATTAC

CATGGTGATG CGGTTTTGGC AGTACATCAA TGGGCGTGGA TAGCGGTTTG ACTCACGGGG

ATTTCCAAGT CTCCACCCCA TTGACGTCAA TGGGAGTTTG TTTTGGCACC AAAATCAACG

GGACTTTCCA AAATGTCGTA ACAACTCCGC CCCATTGACG CAAATGGGCG GTAGGCGTGT

CCATCCACGC TGTTTTGACC TCCATAGAAG ACACCGGGAC CGATCCAGCC TCCGCGGCCG

GGAACGGTGC ATTGGAACGG ACCTGCAGCA CGTGTTGACA ATTAATACGA CTCACTATAG

CCCCGGGCCA GTGTGCTGGA AGTACCGAGC TCGATCGTAC CCAGCTTGGA GGTTGCAGGG

TCGATGCGAC GCAATCGTCC GATCCGGAGC CGGGACTGTC GGGCGTACAC AAATCGCCCG

CAGAAGCGCG GCCGTCTGGA CCGATGGCTG TGTAGAAGTA CTCGCCGATA GTGGAAACCG

ACGGAAGGAG ACAATACCGG AAGGAACCCG CGCTATGACG GCAATAAAAA GACAGAATAA

Nhe I Xba I

2263

2323

2383

2443

2503

2563

2623

2683

2743

2803

2863

2923

2983

3043

3103

3163

3223


8

Cloning into pHook™-3, continued

General Mole-cular BiologyTechniques

For help with DNA ligations, E. coli transformations, restriction enzyme analysis, DNAsequencing, and DNA biochemistry, please see Molecular Cloning: a Laboratory Manualor Current Protocols in Molecular Biology (see References, page 25).

Maintenance ofpHookª-3 andpHookª-3lacZ

To propagate and maintain the pHookª-3 and pHookª-3lacZ vectors, we recommendthat you resuspend each vector in sterile water to prepare a 1 mg/ml stock solution. Storethe stock solutions at -20¡C.

Use the stock solutions to transform the vectors into a recA, endA1 strain such as TOP10,TOP10F«, DH5a, JM109, or equivalent. Select transformants on LB plates containing 25to 50 mg/ml Zeocinª in Low Salt LB (see the next page for a recipe). Be sure to prepare aglycerol stock of each plasmid for long-term storage.

Any E. coli strain that contains the complete Tn5 transposable element (i.e. DH5aF«IQ,SURE, SURE2) encodes the ble (bleomycin) resistance gene. These strains will conferresistance to Zeocinª. For the most efficient selection it is highly recommended that youchoose an E. coli strain that does not contain the Tn5 gene (i.e. TOP10, DH5a, DH10, etc.).

ImportantThis is a nonfusion vector allowing constitutive mammalian expression of the gene ofinterest. Your insert should contain an ATG initiation codon and a Kozak translationinitiation sequence for proper initiation of translation (Kozak, 1987; Kozak, 1991; Kozak,1990). An example of a Kozak consensus sequence is provided below. Please note thatother sequences are possible (see References, page 23), but the A at position -3 and the Gat position +4 are the most critical (shown in bold). The ATG initiation codon is shownunderlined.

ANN ATG G

Your insert should also contain a stop codon for proper expression of your protein inmammalian cells.

NOTE

1. pHookª-3 does not encode the LacZa fragment; therefore, blue/white screening forrecombinants in E. coli cannot be done with this vector.

2. pHookª-3 does not encode the ampicillin resistance gene; therefore, selection forrecombinants can be done only with Zeocinª.

9

Transformation into E. coli

Introduction Ligation mixtures may be transformed into E. coli strains such as TOP10 (Catalog no.C610-00), TOP10F« (Catalog no. C615-00), DH5a, or JM109 (Catalog no. C666-00) andselected on Low Salt LB medium with Zeocinª (see below). Genotypes for these strainsare on the next page. Transformants are isolated and analyzed for the presence andorientation of insert. There is no blue/white screening for the presence of insert withpHookª-3. After obtaining the desired recombinant plasmid, proceed to transfection ofyour mammalian cell line.

IMPORTANT!Low Salt LBMedium withZeocinª

For Zeocinª to be active, the salt concentration of the medium must remain low(<Ê110ÊmM) and the pH must be 7.5. You must prepare LB broth and plates using thefollowing recipe. Please note the lower salt content of this medium.

Failure to lower the salt content of your LB medium will result in non-selectionbecause of inactivation of the drug. Lowering the salt concentration does not affectE.Êcoli growth.

Low Salt LB Medium:

10Êg Tryptone5Êg NaCl5Êg Yeast Extract

1. Combine the dry reagents above and add deionized, distilled water to 950Êml. AdjustpH to 7.5 with 1ÊN NaOH. Bring the volume up to 1 liter. For plates, add 15Êg/L agarbefore autoclaving.

2. Autoclave on liquid cycle at 15Êlbs/sq. in. and 121¡C for 20Êminutes.

3. Allow the medium to cool to at least 55¡C before adding the Zeocinª to 25 µg/mlfinal concentration.

4. Store plates at +4¡C in the dark. Plates containing Zeocinª are stable for 1-2 weeks.

Note: Pre-mixed Low Salt LB Medium is available from Invitrogen in convenient pouchesor in bulk packaging. Please contact Technical Services for more information (see page 23).

Transformation Your construct, pHookª-3 without insert, and the pHookª-3lacZ control vector shouldbe transformed into TOP10F« or similar E. coli strain. Guidelines are as follows:

¥ Transformation may be done by either electroporation or chemical methods.

¥ Add either Low Salt LB or LB medium to the cells after heat shock or electroporationto allow them to recover.

¥ Plate on Low Salt LB medium with 25Êµg/ml Zeocinª.

¥ Incubate overnight at 37¡C.

If you wish, electrocompetent and chemically competent cells are available fromInvitrogen. We recommend TOP10F« or similar as a general strain for transformation.

Catalog no. Description Efficiency AliquotsC3030-01 One Shotª TOP10F« chemically

competent cells1 x 108 21 x 50 µl

C665-03 Chemically competent TOP10F« 1 x 108 5 x 300ÊµlC665-55 Electrocompetent TOP10F« 1 x 109 5 x 80ÊµlC665-11 Electrocompetent TOP10F« 1 x 109 10 x 80Êµl


10

Transformation, continued

Analysis ofTransformants

Plasmid DNA should be isolated from Zeocinª-resistant transformants and analyzed toconfirm the presence and orientation of the desired DNA fragment (see Ausubel, et al.,1990 or Sambrook, et al., 1989).

¥ Select 10-20 transformants and isolate plasmid DNA.¥ Analyze the DNA by restriction mapping or sequencing.

Once you have your insert cloned in the correct orientation, you may test for expressionin mammalian cell lines.

RE

CO

MMENDAT

ION

Sequencing is recommended to confirm the presence of an initiation codon, a Kozaksequence, and/or a stop codon. pHookª sequencing primers are available to sequenceacross the multiple cloning site. Both primers are available from Invitrogen (Catalog no.N680-02 and N681-02, respectively).

Genotypes ofSelected E. coliStrains

DH5a: F- endA1 recA1 hsdR17 (rk-, mk

+) supE44 thi-1 gyrA96 relA1 f80lacZDM15D(lacZYA-argF)U169

JM109: {F«: traD36 proAB+ lacIq lacZDM15} endA1 recA1 hsdR17 (rk-, mk

+) supE44thi-1 gyrA96 relA1 D(lac-proAB)

TOP10: F-, mcrA Æ(mrr-hsdRMS-mcrBC) f80lacZÆM15 ÆlacX74 deoR recA1araD139 Æ(ara-leu)7697 galU galK rpsL endA1 nupG

TOP10F«: F«{lacIq Tn10 (TetR)}mcrA Æ(mrr-hsdRMS-mcrBC) f80lacZÆM15 ÆlacX74deoR recA1 araD139 Æ(ara-leu)7697 galU galK rpsL endA1 nupG

11

Transfection and Cell Selection

Purpose The purpose of this section is to provide guidelines and recommendations for plasmidpreparation and transfection. At this point, you should have a positive clone with yourfragment inserted in the correct orientation in pHookª-3 for expression. The next step isto isolate very clean DNA and transfect your cell line. The method of transfection willdepend on the cell line used. A basic protocol is provided for transfection usingelectroporation and the positive control vector to optimize transfection and selection.

PlasmidPreparation

You may already have a suitable protocol in your laboratory for large-scale plasmidpurification; if not, refer to Current Protocols in Molecular Biology, pp. 9.1.5-9.1.6.Plasmid DNA must be of high quality and free of contaminants for mammaliantransfection. Contaminated DNA can be toxic to many cell lines. We recommend theS.N.A.P.ª Miniprep Kit (10-15 mg DNA, Catalog no. K1900-01) or the S.N.A.P.ª

MidiPrep Kit (10-200 mg DNA, Catalog no. K1910-01) to purify pHookª-3 or yourconstruct for transfection.

Methods ofTransfection

For established cell lines (e.g. HeLa), please consult original references or the supplier ofyour cell line for the optimal method of transfection (i. e. calcium phosphate, DEAE-dextran, liposome-mediated, or electroporation). It is recommended that you followexactly the protocol for your cell line. Pay particular attention to medium requirements,when to pass the cells, and at what dilution to split the cells. Further information isprovided in Current Protocols in Molecular Biology (Reference section, page 25).

There are a variety of methods available for mammalian cell transfection. Invitrogenoffers the Calcium Phosphate Transfection Kit for mammalian cell transfection and thePerFect Lipidª Transfection Kit to optimize lipid-mediated transfection.

Catalog No. Description Quantity

K2780-01 Calcium Phosphate Transfection Kit 75 reactions

K925-01 PerFect Lipidª Transfection Kit 20Êtransfections

RE

CO

MMENDAT

ION

In cases where you do not have a transfection protocol for your cell line, we recommendthat you try electroporation. Electroporation allows synchronous transfection of your cellline which you may find useful for developmental and signaling experiments. We haveincluded a sample electroporation protocol on the next page for your convenience. Pleasenote that you will have to optimize conditions to achieve the maximum transfectionefficiency.


12

Transfection and Cell Selection, continued

Positive Control pHookª-3lacZ (9.9 kb) is provided as a positive control vector for mammaliantransfection and expression. It may be used to optimize transfection conditions for yourcell line. The E.Êcoli gene encoding b-galactosidase is expressed in mammalian cellsusing the CMV immediate-early promoter. Successful transfection and selection willresult in positive b-galactosidase expression in selected cells and can be easily monitoredwith a colorimetric b-galactosidase assay (see page 15). For a map of pHookª-3lacZ, seethe Appendix, page 22. The sequence of the plasmid is available for downloading fromour World Wide Web site (http://www.invitrogen.com) or by contacting TechnicalService (see page 23).

SampleElectroporationProtocol

The following protocol may be used to transfect your cells using electroporation. Thisprotocol has been found to work with a number of cell lines. For settings to electroporateDNA into mammalian cells, please consult the manufacturer's instructions for yourelectroporation device. You may need to optimize electroporation conditions for your cellline (see page 16).

Before starting: Prepare PBS/3ÊmM EDTA (see Recipes, page 19).

Remember to include a "no DNA" negative control.

1. Change medium on the cells 24 hours prior to electroporation.

2. Harvest the cells at 60-80% confluence in half of the initial culture volume of PBS/3ÊmM EDTA (e.g. if your cells are in 10Êml of medium, use 5Êml of PBS/EDTA toharvest the cells).

3. Count cells then centrifuge the cells and resuspend them in complete medium at1ÊxÊ107 cells /ml.

4. Use 1-5 µg of your construct (or pHookª-3lacZ) in a volume of 10Êµl or less.

5. Add plasmid mixture to 200 µl of the cell suspension (2 x 106 cells). Mix gently andtransfer to a chilled electroporation cuvette (0.4 cm gap width).

6. Electroporate your cells using the recommended settings for your electroporationdevice.

7. Transfer electroporated cells to a 60Êmm plate containing 5-7Êml complete medium.Incubate plates in a 37¡C, 5% CO2 incubator for 2-48 hours.


13


Cell SelectionUsing the Capture-Tecª pHookª-3System

Once your cells have been transfected, use the protocol below to isolate the desired cells.We use 2.0 x 106 beads per 60Êmm plate of transfected cells. Your conditions may varydue to the method of transfection and the cell line used. The procedure presented below isprovided as an example. Be sure to use sterile technique when performing the followingsteps.

Preparation ofTransfected Cells

We recommend PBS/3ÊmM EDTA (see Recipes, page 19) and complete medium toharvest your cells. Trypsin/EDTA (0.05% trypsin) may also be used to harvest the cells.Be aware that trypsin may digest the sFv, leading to a decrease in selection efficiency.

1. Remove medium from the cells.

2. Add 3-5Êml of PBS/3ÊmM EDTA to the cells. Incubate cells for 5Êminutes at 37¡Cand then harvest cells. You may also wish to harvest untransfected cells (or the cellsfrom the negative transfection control) to use as a negative control when assayingfor b-galactosidase activity (page 15).

3. Centrifuge cells at 800-1000 x g for 5-10Êminutes at room temperature. Decantsupernatant.

4. Resuspend cells in 1Êml complete medium per 60Êmm plate. Pipet the cells up anddown to break up cell clumps and achieve a single-cell suspension. Proceed toPreparation of Magnetic Beads, Step 4.

Preparation ofMagnetic Beads

Beads must be washed before use to remove the sodium azide.

4. Set up a microcentrifuge tube for each 60Êmm plate of cells.

5. Vortex the magnetic bead slurry to resuspend beads and transfer 10 µl (2.0 x 106

beads) to each microcentrifuge tube.

6. Wash beads by adding 1 ml complete medium to each tube and mix by inversion3Êtimes. Pellet beads with a strong magnet or magnetic stand and pipet or aspirateoff medium. (Magnetic stands are available from Invitrogen. Please see page iv forordering information.)


14


Selection ofTransfected Cells

Once a cell suspension is prepared and the magnetic beads are washed, you are nowready to select transfected cells.

7. Add the 1Êml cell suspension from Step 3, Preparation of Transfected Cells, to atube containing washed Capture-Tecª beads. Incubate 30 minutes at 37¡C on aslow rotator (5-10 revolutions per minute).

8. To select cells, place the tubes containing the bead-cell mixture in a magnetic standand mix for 30 seconds to 1 minute with gentle end-over-end rotation.

9. While the tube is still in contact with the magnet, remove the non-selected cells witha pipet. You may wish to save the non-selected cells for analysis (see next page).

10. Remove the tubes from the magnetic stand and resuspend the beads and cells in1Êml complete medium. Vortex gently to resuspend cells.

11. Pellet beads and bound cells using the magnetic stand and pipet off the supernatant.

12. Repeat Steps 10 and 11 two more times.

13. Resuspend selected cells in 100Êµl complete medium (for pHookª-3lacZ control,use X-gal Reagent, see page 19) and count your cells. Cells are ready to culture oranalyze. For optimization of transfection and selection, proceed to the next page.

ImportantThe magnetic beads cannot be removed efficiently from the cells. The presence of thebeads does not appear to interfere with cell growth after selection.

If you are lysing the cells, the beads may be removed after lysis by using a magnet topellet the beads. The supernatant may then be transferred to a new tube.

15

Optimization of Transfection and Selection

Introduction The pHookª-3lacZ positive control plasmid can be used to check for transfection andselection of cells and to assess transfection efficiencies. Transfected cells are selectedusing the Capture-Tecª pHookª-3 System. Untransfected cells, selected cells, and non-selected cells are assayed with X-gal and counted. Cells expressing b-galactosidase willturn blue in the presence of X-gal. Comparison of the number of blue non-selected cellsversus blue selected cells will allow you to determine the selection efficiency.Untransfected cells should not stain with X-gal.

Before Starting You will need to prepare X-gal Reagent (see Recipes, page 19). This reagent is alsoavailable in the b-Gal Staining Kit (Catalog no. K1465-01) for your convenience. Pleasenote that this reagent will kill the cells. You will not be able to culture the cells afterstaining with X-gal.

Colorimetric Assayfor b-galactosidase

To assay selected cells:

1. Resuspend selected cells (Selection of Transfected Cells, Step 13, page 14) in100Êµl X-gal Reagent.

2. Incubate cells overnight at room temperature.

3. Check the cells under the microscope for the development of blue color. Count totalcells and stained cells.

To assay non-selected cells:

1. Take the non-selected cells obtained from Selection of Transfected Cells, Step 9,page 14 and centrifuge 5 minutes at 4000 rpm to pellet the cells. Decant thesupernatant.

2. Resuspend the cells in 1Êml PBS and pellet the cells. Decant the supernatant.

3. Resuspend the cells in 100Êµl of X-gal Reagent and incubate overnight at roomtemperature.

4. Check the cells under the microscope for the development of blue color. Count totalcells and blue cells.

To assay untransfected cells (negative control):

1. Take the untransfected cells obtained from Preparation of Cells, Step 1, page 13and centrifuge 5 minutes at 4000Êrpm to pellet the cells.

2. Resuspend the cells in 1Êml PBS and pellet the cells.

3. Resuspend the cells in 100Êµl of X-gal Reagent and incubate overnight at roomtemperature.

4. Check the cells under the microscope for the development of blue color. Count totalcells and blue cells.

Remember to normalize to total cell number.


16

Optimization of Transfection and Selection, continued

Optimizationof Selection

When counting your cells after selection, you can easily see the magnetic beads. Youshould see some unbound beads, indicating that the beads are in excess over the numberof transfected cells. This means that you have isolated as many as possible of thetransfected cells that express the sFv in the total population of cells. If you do not see anyunbound beads, it may mean that not all of the transfected cells were selected. You maywish to double the number of beads (20Êµl or 4 x 106 beads) the next time you select cellsto ensure that you isolate all of your transfected cells.

Ideally, nearly all selected cells should express b-galactosidase.

OptimizingElectroporation

All mammalian cell electroporations are generally performed using 0.4Êcm cuvettes. Thevoltage and capacitance must be optimized for each cell line used. The resistance isdetermined by the electroporation buffer and the volume in which the cells aresuspended. Other parameters to optimize are listed below. Start with the first one andproceed through the other parameters until you have increased your transfectionefficiencies. Maximum efficiency of electroporation occurs when the pulse is strongenough to kill 40-80% of your cells. For more information, please see the referenceslisted on page 25-26.

1. Electroporation buffer

¥ "High salt" (sterile PBS without calcium or magnesium cations)

¥ "Low salt" (growth medium without serum supplements)

2. Voltage

¥ For high salt buffer, use 200-1200ÊV

¥ For low salt buffer, use 100-400ÊV

3. Capacitance

¥ For high salt buffer, start at 50 µF and increase to lengthen pulse

¥ For low salt buffer, start at 1000ÊµF and reduce to shorten pulse

4. Volume

Start with 250Êµl and increase to 500Êµl to reduce resistance

Transfection andSelection withYour pHookª-3Construct

After you have optimized transfection and selection with pHookª-3lacZ, transfect yourconstruct into your cell line. Once you have obtained transfected cells, you may analyzethem as desired. It is recommended that you confirm that your gene is expressed in thetransfected cells by using any of the following methods: western blot or enzymatic assay.

17

Technical Assistance

Troubleshooting The table below describes solutions to some possible problems.

Problem Reason Solution

Poor Selection ofTransformants

Too many cells plated. Plate fewer cells or increasethe concentration ofZeocinª to 50Êµg/ml.

Low or No Transfection Method of transfection isnot optimal.

Optimize transfection orchange transfectionmethod.

Low or No Selection Time points of selectionwere too early. Cell linesmay vary in their kineticsof expression

Perform a time course ofselection. Select at 2, 4, 8,16, 24, and 48 hours post-transfection.

Not enough cells Scale-up transfection andplate in 100Êmm plates

sFv is not expressed Use antibody to either themyc or Hemagglutinin Aepitopes to detect the sFvprotein in a western blot.

sFv is not displayed You may wish to test cellsusing in situ immunofluor-escence with antibodies tothe Hemagglutinin A ormyc epitopes.

Technical Service For Technical Service, please call, write, fax or E-mail:


18

Appendix

Recipes

Low Salt LB AgarPlates withZeocinª

Low Salt LB Medium (per liter)

1% Tryptone0.5% Yeast Extract0.5% NaCl1.5% Agar

pH 7.0

1. For 1Êliter, dissolve 10Êg tryptone, 5Êg yeast extract, and 5Êg NaCl in 950Êmldeionized water.

2. Adjust the pH of the solution to 7.5 with 5ÊM NaOH, add 15Êg agar, and bring thevolume to 1Êliter.

3. Autoclave for 20Êminutes on liquid cycle.

4. Let agar cool to ~55¡C. Thaw the 100Êmg/ml Zeocinª stock solution and add to afinal concentration of 25Êµg/ml (250Êµl/liter of medium).

Sufficient Zeocinª is provided to make 4Êliters of 25Êµg/ml Zeocinª medium.

5. Pour into 10Êcm petri plates. Let the plates harden, then invert and store at +4¡C.Plates containing Zeocinª is stable for 1-2 weeks.

YT-Zeocinª Plates YT medium (per liter)

0.8% Tryptone0.5% Yeast extract0.25% NaCl1.5% agar

1. For 1Êliter, dissolve 8Êg tryptone, 5Êg yeast extract, and 2.5Êg NaCl in 900Êmldeionized water.

2. Adjust pH to 7.5 with 5ÊM NaOH, add 15Êg agar, and bring up the volume to 1 liter.

3. Autoclave for 20Êminutes on liquid cycle.

4. Cool to the medium to ~55¡C. Thaw the 100Êmg/ml Zeocinª stock solution.

5. Add Zeocinª to 25Êµg/ml final concentration (250Êµl).


19

Recipes, continued

PhosphateBuffered Saline(with EDTA)

Composition

137ÊmM NaCl2.7ÊmM KCl10ÊmM Na2HPO4

1.8ÊmM KH2PO4

(3ÊmM EDTA, optional)

1. Dissolve the following in 800Êml deionized water:

8Êg NaCl0.2Êg KCl1.44Êg Na2HPO4

0.24Êg KH2PO4

(6Êml 0.5ÊM EDTA, pH 8)

2. Adjust pH to 7.4 with concentrated HCl.

3. Bring the volume to 1Êliter and autoclave for 20 minutes on liquid cycle.

4. Store at +4¡C or room temperature.

X-Gal Reagent forStaining Cells

Composition

1 mg/ml X-Gal in DMF4 mM potassium ferricyanide (K3Fe(CN)6)4 mM potassium ferrocyanide (K4Fe(CN)6-3H2O)2 mM magnesium chloride hexahydrate

in PBS, pH 7.4

Prepare just enough solution to stain the cells you desire.

1. Prepare 10Êml each of the following stock solutions. Solutions are stable indefinitelyif stored as indicated.

¥ X-gal: 20 mg/ml in dimethylformamide (DMF) Dissolve 200 mg in 10 ml DMFand store at -20¡C.

¥ Potassium Ferricyanide and Potassium Ferrocyanide: 0.4ÊM each in deionizedwater. Dissolve 1.32Êg of potassium ferricyanide and 1.69Êg of potassiumferrocyanide in 10Êml deionized water. Store at -20¡C.

¥ Magnesium Chloride: 200ÊmM in deionized water. Dissolve 0.4Êg in 10Êmldeionized water and store at room temperature or -20¡C.

2. For 10Êml of reagent, mix together:

0.5Êml of 20Êmg/ml X-Gal stock solution0.1Êml of the potassium ferricyanide/ferrocyanide stock solution0.1Êml of the magnesium chloride stock solution9.3Êml of PBS

3. Solution is ready for use. Please note that this solution will kill the cells.

Note: If you wish, there is a b-Gal Staining Kit available from Invitrogen (Catalog no.K1465-01). The kit contains everything you need to fix and stain cells for b-galactosidaseactivity. Please call Technical Service for more information (see page 23).

20

Zeocin™

Description Zeocinª belongs to a family of structurally related bleomycin/phleomycin-typeantibiotics isolated from Streptomyces. Antibiotics in this family are broad spectrumantibiotics that act as strong antibacterial and antitumor drugs. They show strong toxicityagainst bacteria, fungi (including yeast), plants, and mammalian cells. Zeocinª is not astoxic as bleomycin on fungi. As a broad-spectrum antibiotic Zeocinª is particularlyuseful, allowing selection in a number of cell types containing vectors with a Zeocinª

resistance gene.

ChemicalProperties

Zeocinª is a basic, water-soluble compound isolated from Streptomyces verticillus as acopper-chelated glycopeptide. The presence of copper gives the solution its blue color.The chemical formula for Zeocinª is C55H83N19O21S2Cu. It contains several uniqueamino acids, sugars, and aliphatic amines. For general information about the family ofbleomycin antibiotics, please see (Berdy, 1980) (Reference section, page 25). Thegeneral structure of Zeocinª is shown below.

CH3

HO

O

CH3 HO

O R

O

CH3

O

CH3H2N

OH

H

O

H2N

CONH2H

O

OHHO HO

HO

OH

OH

R =

NH

HNNH2

HN

++Cu

H2N

N N

N

N

N

NH2

O

O

O

O

O

HN N

N

S

NH

HN

NH N

S

Mechanism ofAction

The exact mechanism of action of Zeocinª is not known; however, it is thought to be thesame as bleomycin and phleomycin due to its similarity to these drugs and its inhibitionby the Sh ble resistance protein (see next section). The copper/glycopeptide complex isselective and involves chelation of copper (Cu2+) by the amino group of the a-carbox-amide, single nitrogen atoms of both the pyrimidine chromophore and the imidazolemoiety, and the carbamoyl group of mannose. The copper-chelated form is inactive.When the antibiotic enters the cell, the copper cation is reduced from Cu2+ to Cu+ andremoved by sulfhydryl compounds in the cell. Upon removal of the copper, Zeocinª isactivated to bind DNA and cleave it causing cell death (Berdy, 1980). Saltconcentrations greater than 110ÊmM and acidity or basicity inactivate Zeocinª;therefore, it is necessary to reduce the salt in bacterial medium and adjust the pH to7.5 to make sure the drug remains active.


21

Zeocin™, continued

Resistance toZeocinª

A Zeocinª resistance protein has been isolated and characterized (Gatignol, et al., 1988;Drocourt, et al., 1990). This protein, the product of the Sh ble gene (Streptoalloteichushindustanus bleomycin gene), is a 13,665 D protein which binds Zeocinª in a stoichio-metric manner. The binding of Zeocinª inhibits its DNA strand cleavage activity.Expression of this protein in eukaryotic and prokaryotic hosts confers resistance toZeocinª. The nucleic acid and protein sequence is given below:

L R D P A G N C V H F VCTG CGC GAC CCG GCC GGC AAC TGC GTG CAC TTC GTG

T N F R D A S G P A M T

D N T L A W V W V R G L

ACG AAC TTC CGG GAC GCC TCC GGG CCG GCC ATG ACC

GAC AAC ACC CTG GCC TGG GTG TGG GTG CGC GGC CTG

D D F A G V V R D D V TGAC GAC TTC GCC GGT GTG GTC CGG GAC GAC GTG ACC

T A R D V A G A V E F WACC GCG CGC GAC GTC GCC GGA GCG GTC GAG TTC TGG

GCC GAG GAG CAG GAC TGA

Nco I

Sma I

SgrA I

A E E Q D STOP

ACC ATG GCC AAG TTG ACC AGT GCC GTT CCG GTG CTC M A K L T S A V P V L

ACC GAC CGG CTC GGG TTC TCC CGG GAC TTC GTG GAG T D R L G F S R D F V E

E I G E Q P W G R E F A

CTG TTC ATC AGC GCG GTC CAG GAC CAG GTG GTG CCG

GAC GAG CTG TAC GCC GAG TGG TCG GAG GTC GTG TCC

GAG ATC GGC GAG CAG CCG TGG GGG CGG GAG TTC GCC

L F I S A V Q D Q V V P

D E L Y A E W S E V V S

22

pHook™-3lacZ

Description pHookª-3lacZ is a 9907 bp control vector containing the gene for b-galactosidase. Thisvector was constructed by ligating a 3.1 kb fragment containing the lacZ gene intopHookª-3lacZ.

Map of ControlVector

The figure below summarizes the features of the pHookª-3lacZ vector. The completenucleotide sequence for pHookª-3lacZ is available for downloading from ourWorld Wide Web site (http://www.invitrogen.com) or by contacting TechnicalService (see next page).

Comments for pHook™-3 lacZ 9907 nucleotides

SV40 origin and promoter: bases 6-785EM-7 promoter: bases 776-853Sh ble gene (ZeoR): bases 854-1228Early SV40 polyadenylation signal: bases 1292-1421pMB1 (pUC-derived) origin: bases 1531-2204CMV promoter: bases 2263-2881pHook™-3 Forward priming site: bases: 2866-2884LacZ ORF: 3015-6270pHook™ Reverse priming site: bases: 6748-6766TK polyadenylation signal: bases 6906-7274RSV LTR: bases 7711-8063Murine Ig kappa-chain V-J2-C signal peptide: bases 8074-8136Hemagglutinin A epitope: bases 8137-8163phOx sFv: bases 8179-8892myc epitope 1: bases 8902-8931myc epitope 2: bases 8947-8976PDGFR transmembrane domain: bases 8980-9129BGH polyadenylation signal: bases 9191-9419f1 origin: bases 9482-9895

SignalPeptide


A

myc

mycPCMV PRSVTKpA

pMB1 ori BGH pA

SV

40

Hin

d III

Zeocin EM-7

f1oripHook ™-3/

lacZ9.9 kb

SV40

pAN

he I

Xba

I

lacZ

23

Technical Service

World Wide Web Visit the Invitrogen Web Resource using your World Wide Web browser. At the site,you can:

¥ Get the scoop on our hot new products and special product offers¥ View and download vector maps and sequences¥ Download manuals in Adobeª Acrobatª (PDF) format¥ Explore our catalog with full color graphics¥ Obtain citations for Invitrogen products¥ Post a question at one of our many user forums¥ Request catalog and product literature

Once connected to the Internet, launch your web browser (Netscape 3.0 or newer), thenenter the following location (or URL):

http://www.invitrogen.com

...and the program will connect directly. Click on underlined text or outlined graphics toexplore. Don't forget to put a bookmark at our site for easy reference!

Phone and E-mail If you need technical information or help, please E-mail, call, or fax us:

Location E-mail Telephone/Fax

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United States

[email protected] Voice: 1-800-955-6288 (free)

Fax: 1-760-603-7201

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Addresses U.S. Headquarters: European Headquarters:Invitrogen Corporation Invitrogen BV1600 Faraday Avenue PO Box 2312, 9704 CH GroningenCarlsbad, CA 92008 USA The Netherlands


24

Technical Service, continued

Limited Warranty Invitrogen is committed to providing our customers with high-quality goods and services. Our goal is toensure that every customer is 100% satisfied with our products and our service. If you should have anyquestions or concerns about an Invitrogen product or service, please contact our Technical ServiceRepresentatives at:

1-800-955-6288, extension 2 (U.S. and Canada)

00800 5345 5345 (toll free) or +31 (0) 50 5299 299 (Europe, Middle East, and Africa)

1-760-603-7200 extension 2 (all others)

Invitrogen warrants that all of its products will perform according to the specifications stated on the certificateof analysis. The company will replace, free of charge, any product that does not meet those specifications. This warranty limits Invitrogen CorporationÕs liability only to the cost of the product . No warranty is grantedfor products beyond their listed expiration date. No warranty is applicable unless all product components arestored in accordance with instructions. Invitrogen reserves the right to select the method(s) used to analyze aproduct unless Invitrogen agrees to a specified method in writing prior to acceptance of the order.

Invitrogen makes every effort to ensure the accuracy of its publications, but realizes that the occasionaltypographical or other error is inevitable. Therefore Invitrogen makes no warranty of any kind regarding thecontents of any publications or documentation. If you discover an error in any of our publications, pleasereport it to our Technical Service Representatives.

Invitrogen assumes no responsibility or liability for any special, incidental, indirect or consequentialloss or damage whatsoever. The above limited warranty is sole and exclusive. No other warranty ismade, whether expressed or implied, including any warranty of merchantability or fitness for aparticular purpose.

25

References

General MolecularBiologyTechniques

Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A. andStruhl, K., eds (1990) Current Protocols in Molecular Biology. Greene PublishingAssociates and Wiley-Interscience, New York.

Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A LaboratoryManual, second edition, Cold Spring Harbor Laboratory Press, Plainview, NewYork.

MammalianTransfection andSelection

Baum, C., Forster, P., Hegewisch-Becker, S. and Harbers, K. (1994) An OptimizedElectroporation Protocol Applicable to a Wide Range of Cell Lines. BioTechniques17: 1058-1062.

Berdy, J. (1980) Bleomycin-Type Antibiotics. In: J. Berdy (ed) Handbook of AntibioticCompounds. Vol. IV, Part I. Amino Acid and Peptide Antibiotics. CRC Press. BocaRaton, FL. pp. 459-497.

Boshart, M., Weber, F., Jahn, G., Dorsch-Hasler, K., Fleckenstein, B. and Schaffner, W.(1985) A Very Strong Enhancer Is Located Upstream of an Immediate Early Geneof Human Cytomegalovirus. Cell 41: 521-530.

Chesnut, J. D., Baytan, A. R., Russell, M., Chang, M.P., Bernard, A., Maxwell, I. H. andHoeffler, J. P. (1996) Selective Isolation of Transiently Transfected Cells from aMammalian Cell Population with Vectors Expressing a Membrane AnchoredSingle-Chain Antibody. J. Imm. Methods 193: 17-27.

Chen, C. and Okayama, H. (1987) High-Efficiency Transformation of Mammalian Cellsby Plasmid DNA. Molec. Cell. Biol. 7: 2745-2752.

Chu, G., Hayakawa, H. and Berg, P. (1987) Electroporation for the Efficient Transfectionof Mammalian Cells with DNA. Nucleic Acids Res. 15: 1311-1326.

Coloma, M.J., Hastings, A., Wims, L.A. and Morrison, S.L. (1992) Novel Vectors for theExpression of Antibody Molecules Using Variable Regions Generated byPolymerase Chain Reaction. J. Imm. Methods 152: 89-104.

Drocourt, D., Calmels, T. P. G., Reynes, J. P., Baron, M. and Tiraby, G. (1990) Cassettesof the Streptoalloteichus hindustanus ble Gene for Transformation of Lower andHigher Eukaryotes to Phleomycin Resistance. Nucleic Acids Res. 18: 4009.

Evans, G.I., Lewis, G.K., Ramsay, G. and Bishop, V.M. (1985) Isolation of MonoclonalAntibodies Specific for c-myc Proto-oncogene Product. Mol Cell. Biol. 5: 3610-3616.

Felgner, P.L., Gadek, T.R., Holm, M., Roman, R., Chan, H.W., Wenz, M., Northrop, J.P.,Ringold, G.M. and Danielsen, M. (1987) Lipofectin: A Highly Efficient, Lipid-Mediated DNA-Transfection Procedure. Proc. Natl. Acad. Sci. USA 84: 7413-7417.

Gatignol, A., Durand, H. and Tiraby, G. (1988) Bleomycin Resistance Conferred by aDrug-binding Protein. FEB Letters 230: 171-175.

Goodwin, E.C. and Rottman, F.M. (1992) The 3« Flanking Sequence of the BovineGrowth Hormone Gene Contains Novel Elements Required for Efficient andAccurate Polyadenylation. J. Biol. Chem. 267: 16330-16334.

Gorman, C.M., Merlino, G.T., Willingham, M.C., Pastan, I. and Howard, B.H. (1982)The Rous sarcoma virus long terminal repeat is a strong promoter when introducedinto a variety of eukaryotic cells by DNA-mediated transfection. Proc. Natl. Acad.Sci. USA 79: 6777-7681.


26

References, continued

MammalianTransfection andSelection,continued

Griffiths, G.M., Berek, C., Kaartinen, M. and Milstein, C. (1984) Somatic Mutation andthe Maturation of Immune Response to 2-phenyl-oxazolone. Nature 312: 271-275.

Gronwald, R.G., Grant, F.J., Haldeman, B.A., Hart, C.E., O'Hara, P.J., Hagen, F.S., Ross,R., Bowen-Pope, D.F. and Murray, M.J. (1988) Cloning and Expression of a cDNACoding for the Human Platelet-Derived Growth Factor Receptor: Evidence forMore Than One Receptor Class. Proc. Natl. Acad. Sci. USA 85: 3435-3439.

Hoogenboom, H.R., Griffiths, A.D., Johnson, K.S., Chriswell, D.J., Hudson, P. andWinter, G. (1991) Multi-subunit Proteins on the Surface of Filamentous Phage:Methodologies for Displaying Antibody (Fab) Heavy and Light Chains. Nucl. AcidsRes. 19: 4133-4137.

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