25-8010-20 akt1-egfp assay...25-8010-17um, page finder, rev a, 2003 um front cover: top image:...

user manual25-8010-1725-8010-1825-8010-1925-8010-20

AKT1-EGFP Assay

25-8010-17UM,

Rev-A, 2003

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user manual

25-8010-1725-8010-1825-8010-1925-8010-20

AKT1-EGFP Assay

25-8010-17UM

Rev-A, 2003

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user manual

25-8010-1725-8010-1825-8010-1925-8010-20

AKT1-EGFP Assay

25-8010-17UM,

Rev A, 2003

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Front cover:

Top image: CHO-hIR cells containing theAKT1-EGFP fusion protein before theaddition of agonist.

Bottom image: The same field of view, 4 min after stimulation with agonist (2 µg/ml IGF-1). Images shown are1/20th of the actual image size acquiredby the IN Cell Analyzer 3000.

BioImage is a Danish Biotech companyspecializing in developing drugcandidates that exert their activitythrough modulation of proteintranslocation. For more information, visittheir Web site at www.bioimage.dk

Page finder

Chapter 1. Introduction1.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

1.2. AKT1-EGFP assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

Chapter 2. Licensing Considerations2.1. Right to use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

2.2. Legal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

Chapter 3. Product Contents3.1. Components summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

3.2. CHO derived cell line expressing AKT1-EGFP fusion protein – NIF2019. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

3.2.1. CHO derived parental cell line . . . . . . . . . . . . . . . . . . . . . . . . . . 1

3.2.2. CHO derived AKT1-EGFP expressing cell line . . . . . . . . . . . . . . . . 1

3.3. AKT1-EGFP expression vector – NIF2020. . . . . . . . . . . . . . . . . . . . 1

3.4. Materials and equipment required . . . . . . . . . . . . . . . . . . . . . . . . 2

3.5. IN Cell Analysis System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

3.5.1. IN Cell Analyzer 3000. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

3.5.2. Plasma Membrane Spot analysis Module . . . . . . . . . . . . . . . . . . . 3

3.5.3. IN Cell Analyzer 1000. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

3.6. AKT1-EGFP translocation assay on epifluorescence microscopes . . . 3

3.7. Software requirements. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Chapter 4. Safety Warnings, Handling andPrecautions 4.1 Safety warnings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

4.2. Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

4.3. Handling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

4.3.1. Vector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

4.3.2. Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

Chapter 5. Cell Assay Design 5.1. Culture and maintenance of CHO derived AKT1-EGFP

expressing cell line . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

5.1.1. Tissue culture media and reagents required . . . . . . . . . . . . . . . . . 1

5.1.2. Reagent preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

5.1.3. Cell thawing procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

5.1.4. Cell sub-culturing procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

5.1.5. Cell seeding procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

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5.1.6. Cell freezing procedure. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

5.1.7. Growth characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

5.2. Assay set up. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

5.2.1. Live cell AKT1-EGFP assay using the IN Cell Analyzer 3000 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

5.2.2. Microplate set up for 96 well format assays . . . . . . . . . . . . . . . . . 4

5.2.3. Schematic agonist assay protocol . . . . . . . . . . . . . . . . . . . . . . . . 5

5.2.4. Antagonist assay protocol (96 well format) . . . . . . . . . . . . . . . . . . 5

5.2.5. Schematic antagonist assay protocol . . . . . . . . . . . . . . . . . . . . . . 6

5.2.6. Antagonist assay protocol (96 well format) . . . . . . . . . . . . . . . . . . 7

5.2.7. Timing schedule. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

5.2.8. Important considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

5.2.9. Fixed cell assay format . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

5.3. Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

5.3.1. Calculating Z’-factor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

5.3.2. Example results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

5.4. Assay characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

5.4.1. Translocation index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

5.4.2. Summary of quantitative assay parameters. . . . . . . . . . . . . . . . . 11

5.4.3. Seeding density . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

5.4.4. IGF-1 dose response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

5.4.5. Wortmannin inhibition curve . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

5.4.6. IGF-1 agonist time course . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

5.4.7. Sensitivity of assay to DMSO, Ethanol and Methanol . . . . . . . . . . 14

5.4.8. Effects of different assay-medium . . . . . . . . . . . . . . . . . . . . . . . 15

5.4.9. Effect of serum starvation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

5.4.10. Fixed assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

5.4.11. Insulin agonist . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

5.4.12. DRAQ5 nuclear stain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

5.4.13. Results obtained fron the IN Cell Analyzer 1000 . . . . . . . . . . . . . 19

Chapter 6. Vector use details 6.1. General guidelines for vector use . . . . . . . . . . . . . . . . . . . . . . . . . 1

6.2. Transient transfection with pCORON1000 AKT1-EGFP . . . . . . . . . . . 1

6.3. Stable cell line generation with pCORON1000 AKT1-EGFP . . . . . . . . 1

Chapter 7. Quality Control 7.1. AKT1-EGFP cell line . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

7.2. AKT1-EGFP expression vector. . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

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Chapter 8. Troubleshooting Guide 8.1. Troubleshooting guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Chapter 9. References 9.1. References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Chapter 10. Related Products 10.1. Related products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Chapter 11. Appendix 11.1. Appendix A: Restriction map of pCORON1000 AKT1-EGFP . . . . . . . . 1

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Chapter 1. Introduction

1.1 IntroductionAKT1, (also known as PKBα or RAC-Pkα) is a serine/threonine kinase thatmediates a number of intracellular pathways, including cell survival andinsulin signalling (Fig 1.2, for reviews see 1 and 2). Activation of AKT1provides a strong survival signal that protects cells from apoptosis(programmed cell death). The AKT1 oncogene has attracted much attention asa drug target because of its central role in regulating cell proliferation andsurvival (for review see 3). An increasing number of AKT substrates have beenimplicated in oncogenesis (2). AKT1 kinase activity is elevated in prostate,breast and ovarian cancers (4). Elevated AKT1 kinase activity has also beencorrelated with poor prognosis of cancers, and has been implicated inresistance to tamoxifen and radiation therapies (5, 6). AKT1 has been foundto be amplified in at least one type of cancer (7), and elevated expression ofAKT1 has been implicated in angiogenesis, which is an important factor fortumor progression (8).

Structure and functional domainsAKT1 has an amino-terminal pleckstrin homology (PH) domain, a centralkinase domain, and a carboxy-terminal hydrophobic and proline-rich domain(Fig 1.1). Two other family members with a high degree of sequence homologyto AKT1 have been cloned-AKT2/PKBß/RAC-PKß and AKT3/RAC-PKγ.Alignment of the gene sequences from AKT family members suggests that theprimary structure is evolutionarily conserved, with the exception of thecarboxy-terminal tail, which is found in some but not all species and isoforms(2). The PH domain of AKT1 plays an essential role in both its localizationand its activation.

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Fig 1.1. AKT1 signalling pathway(provided with permission fromBioCarta), (www.biocarta.com).

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Mode of actionDepending on the physiological context, AKT1 can be activated by a range ofmitogenic factors, including IGF-1, PDGF, insulin, thrombin and NGF. AKT1is regulated by a class Ia PI3-Kinase, and PI3-Kinase activity has beenshown to be necessary for AKT1 activation (Fig 1.2). Growth factorstimulation of cells results in activation of Ras and subsequent interaction ofactivated Ras with the p110 catalytic subunit of PI3-Kinase. PI(3,4)P2phosphoinositide generated by PI3-Kinase provides a specific, high-affinitybinding site for the PH domain of AKT1. In response to mitogenic stimuli,AKT1 translocates from the cytosol of the cell to the plasma membrane. Thebinding of PI3-Kinase-generated phosphoinositide headgroups at the plasmamembrane is critical to the relocalization of AKT, which in turn plays animportant role in the process of AKT activation (9). Phosphorylation of AKT1occurs on two sites-Thr308 and Ser473. Two kinases are primarilyresponsible for this phosphorylation event. These are PDK1 (PIP3-dependentkinase 1) and PDK2. PDK1 phosphorylates Thr308, and is stimulated byPI3-Kinase via PIP3 and PIP2. PDK2 or PDK1 in conjunction with anotherkinase (PRK-2) phosphorylates Ser473, again controlled by PI3-Kinase. Themembrane localization and subsequent phosphorylation of AKT1 at Thr308and Ser473 are necessary for full activation of AKT1 kinase activity (10, 11,12). The activation of AKT1 is negatively regulated by the phosphatasesPTEN and SHIP, which reduce the level of PIP3 in the plasma membrane(2).

AKT1 substratesThe preferred substrate of AKT1 in vitro is RxTyz(S/T)(hy) where x is anyamino acid, y and z are small residues other than glycine and hy is a bulkyhydrophobic group. However it is important to consider that in vivo thesubstrate specificity of AKT may partially depend on sequence determinantsthat lie outside the consensus sequence (13, 14). There are a large numberof substrates phosphorylated by AKT1 and these are linked with severaldifferent intracellular pathways, including cell cycle regulation,differentiation, glycolysis, GLUT 4 translocation, protein synthesis and thesurvival/apoptosis pathways.

ApoptosisOne important function of the AKT1 signalling pathway is to promote growthfactor-mediated cell survival and protect cells from programmed cell death(apoptosis). The identification of the AKT1 consensus phosphorylationsequence in proteins involved in the apoptotic process indicated that AKT1regulates cell survival by directly phosphorylating components of the celldeath machinery. Various Bcl-2 family members are part of a network ofapoptotic regulators that are located both in the cytoplasm and inintracellular membranes (15). In the presence of survival factors, the activityof pro-survival Bcl-2 family members prevails over the activity of pro-death

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Fig 1.2 Domain structure of humanAKT1.


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Bcl-2 family members, and apoptosis is inhibited. Initial studies on AKT1’sinvolvement in apoptosis were performed on C.elegans and the Bcl-2 relatedproteins ced-9 and egl-1. Activated AKT1 phosphorylates and deactivates thepro-death Bcl-2 family member BAD (Ser136). Phosphorylated BAD binds 14-3-3, which protects it from dephosphorylation, and thus inhibits apoptosis (16,17). Caspase 9 proteolytic activity cleaves a variety of cellular substrates thatare critical to the generation of the apoptotic phenotype. Inhibition of caspase9 by AKT-mediated phosphorylation prevents this phenotype evolving (18).

Prolonged activation of AKT1 (e.g. 30 min) causes AKT1 to localize from thecytoplasm to the nucleus where it has been hypothesized to phosphorylate andmodulate the activity of several transcription factors (9). In mammalian cells,three members of the Forkhead family of transcriptional activators have beenidentified in certain human tumors, FKHR, FKHRL1/AF6q21, and AFX. Eachof these proteins has a Forkhead domain that mediates interaction with DNA.Forkhead proteins also have AKT phosphorylation sites. AKT-mediatedphosphorylation modulates the function of Forkhead by regulating its cellularlocation. Phosphorylated Forkhead is retained in the cytoplasm and is inactive.Non-phosphorylated Forkhead is transcriptionally active. AKT promotes cellsurvival by phosphorylating and sequestering Forkhead transcription factors inthe cytoplasm, thereby preventing them from inducing the transcription ofcritical death genes (19, 20).

Recent evidence has implicated AKT as a signalling intermediate upstream ofsurvival gene expression that is dependent on the transcription factor NFκB.NFκB is sequestered in the cytoplasm by proteins of the IκB family (21).Phosphorylation of IκB targets it for ubiquitinylation and degradation.Degradation of IκB frees NFκB, allowing its nuclear translocation andactivation of target genes. IκB is regulated by a protein complex that includestwo kinases IKKα and IKKß, both of which are capable of phosphorylating IκB.AKT has been shown to enhance the degradation of the IκBs, possibly throughactivation of one of the IKKs, and to co-operate with other factors to induceNFκB responsive model promoters (22).

MetabolismAKT1 is integrally involved in cellular processes regulating several metabolicenzymes. Phosphorylation of GSK-3 (glycogen synthase kinase 3) at Ser9results in its inactivation and activation of glycogen synthesis. Recently, GSK-3has been implicated as a mediator of the PI3-Kinase survival signal.Constitutively active GSK-3 induces apoptosis even in the presence of growthfactors. The reason for this is still unclear (23).

AKT1 phopshorylates and regulates p70S6kinase (protein synthesis) (24); AKT1stimulates glucose uptake and GLUT 4 translocation (25) and AKT1 inducesadipocyte differentiation in 3T3-L1 cells (26). Phosphorylation of E2F byAKT1 results in cell cycle regulation (27). 6-phosphofructo-2-kinase (PFK-2)phosphorylation of AKT1 gives an increase in glycolysis (28). Cellular stressessuch as heat shock and hyperosmolality also stimulate AKT1. It is postulatedthat this is through the activation of the p38/HOG1 kinase cascade (29). Theprecise role of AKT in stress signalling is unknown. A number of additionalsubstrates for AKT1 have been identified including eNOS, the activation ofwhich influences long-term blood vessel growth, and the reverse transcriptasesubunit of telomerase, which is responsible for telomere maintenance andDNA stability (30, 31).


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Agonist,4 min

Un-stimulated cell,Majority of AKT1-EGFP

localized in cytoplasmic space

Stimulated cell,AKT1-EGFP redistributes to

plasma membrane

Fig 1.3. Agonist-induced redistributionof AKT1-EGFP from the cytoplasm toplasma membrane.

1.2 AKT1-EGFP assayBecause of its involvement in numerous intracellular pathways, AKT1 ispotentially an excellent drug target for therapeutic treatment of numerousdisease states, particularly cancer therapy. A live-cell screening assayexamining the AKT1 signalling pathway has been developed (Fig 1.3). Theassay uses Redistribution™ technology to quantify the intracellulartranslocation of an AKT1-EGFP fusion protein in a stably transfectedmammalian cell line. Upon activation of the AKT1 pathway, AKT1translocates from the cytosol to the plasma membrane and its PH domainbinds to phospholipids in the plasma membrane. The AKT1 Redistribution™assay monitors redistribution of AKT1-EGFP fusion protein from thecytoplasm to the plasma membrane in cells challenged with testcompounds. IGF-1 can be used as a reference agonist (EC50 typically 23ng/ml). Wortmannin can be used as an inhibitor of the AKT1-EGFP fusionprotein (IC50 typically 15.8 nM).

This assay is optimized for image acquisition and analysis on the IN CellAnalyzer 3000 using the Plasma Membrane Spot analysis module, althoughthe assay can also be imaged on other systems.


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Chapter 2. Licensingconsiderations 2.1. Right to useUse of this assay is limited as stated in the terms and conditions of sale.These vary in accordance with the product code purchased.

Description Product CodeAKT-1-EGFP Assay, Screening applications 25-8010-17AKT-1-EGFP Assay, Research applications 25-8010-18AKT-1-EGFP Assay, 6 month assay evaluation 25-8010-19AKT-1-EGFP Assay, 12 month assay evaluation 25-8010-20

This assay was developed in collaboration with BioImage A/S and is soldunder license from:

BioImage A/S under patents US 6172188, US 5958713, US6518021, EP851874, EP 0815257,EP 0986753 and other pending and foreign patentapplications; and Invitrogen IP Holdings Inc (formerly Aurora Biosciences Corporation) underUS patents: US 5625048, 5777079, 5804387, 5968738, 5994077,6054321, 6066476, 6077707, 6090919, 6124128, 6319969, 6403374European Patent 1104769, 0804457 and Japanese patent JP 3283523and other pending and foreign patent applications; and Columbia University. This product is also sold under license from ColumbiaUniversity under US patent numbers 5491084 and 6146826. Rights to usethis product, as configured, are limited to internal use for screening,development and discovery of therapeutic products; NOT FOR DIAGNOSTICUSE OR THERAPEUTIC USE IN HUMANS OR ANIMALS. No other rights areconveyed; andUniversity of Florida Research Foundation under patents US 5968750,5874304, 5795737, 6020192 and other pending and foreign patentapplications; andIowa Research Foundation. The CMV promoter is covered under US patents5168062 and 5385839 and its use is permitted for research purposes only.Any other use of the CMV promoter requires a license from the University ofIowa Research Foundation 214 Technology Innovation Center Iowa CityIA52242 USA; andFor customers wishing to use the assay for screening for potentialtherapeutic agents attention is drawn to the existence of US Patent Number6 054 280 ‘Methods for Diagnosis and Treatment of PH Domain SignalTransduction Disorders’ issued 25 April 2000 and assigned to Sugen Inc, CAUSA.

The exact terms of use for the product as configured are specified in theTerms and Conditions of Sale accompanying the product, but are limited tointernal use for development and discovery of therapeutic products. Norights other than those expressly granted are conveyed.

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2.2. Legal Cy is a trademarks of Amersham Biosciences Limited

Amersham and Amersham Biosciences are trademarks of Amersham plc

BioImage and Redistribution are trademarks of BioImage A/S

Biocarta is a trademark of Biocarta Inc

FuGENE is a trademark of Fugent, LLC

Microsoft is a trademark of Microsoft Corporation

Hoechst is a trademark of Aventis

Geneticin is a trademark of Life Technologies Inc

DRAQ5 is a trademark of Biostatus Limited

All goods and services are sold subject to Terms and Conditions of Sale ofthe company within the Amersham Biosciences group, which supplies them.Copies of these terms and conditions are available on request.

© Amersham Biosciences UK Limited 2003 - All rights reserved

http://www.amersham.com

Amersham Biosciences UK Limited, Amersham Place Little Chalfont Buckinghamshire HP7 9NA UK

Amersham Biosciences AB, SE-751 84 Uppsala Sweden

Amersham Biosciences Corp, 800 Centennial Avenue PO Box 1327 Piscataway NJ08855 USA

Amersham Biosciences Europe GmbH, Munzinger Strasse 9 D-79111 Freiburg Germany

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Chapter 3. Product contents

3.1. Components summary● CHO-hIR derived cells expressing the AKT1-EGFP fusion protein

(two vials each containing 1 ml and 1 x 106 cells) – NIF2019

● pCORON1000 AKT1-EGFP expression vector (one vial containing 10 µgDNA, at 250 µg/ml, supplied in TE buffer: 10 mM Tris, 1 mM EDTA pH8.0) – NIF2020

● User manual

3.2. CHO-derived cell line expressing AKT1-EGFPfusion protein - NIF2019

3.2.1. CHO-derived parental cell lineThe CHO-hIR cell line is of Chinese hamster ovary origin, derived from CHO-K1 (ATCC CCL-61) cells (32, 33, 34) that have been stably transfectedwith a non-mutated full-length human Insulin receptor (hIR) (35). The cellswere transfected by a non-viral method and the hIR expression is undercontrol of the metallothionein promoter. The vector contains the DHFR genethat allows selection of expressing cells with methotrexate (MTX). The hIR expression appears to be extremely stable in CHO cells withoutselection pressure, the cell line having retained insulin-sensitivity for severalpassages in the absence of MTX.

3.2.2. CHO-derived AKT1-EGFP expressing cell lineCHO-hIR cells were transfected with the pCORON1000 AKT1-EGFP vector(supplied) using FuGENE 6. A stable clone expressing the recombinantfusion protein was selected using 500 µg/ml Geneticin. The isolated clonewas chosen, grown for 12 passages and then frozen. The cells testednegative for mycoplasma, bacterial and yeast contamination (testing detailsare available upon request). MTX selection pressure is not recommendedwith this particular cell line because CHO cells may develop MTX resistanceby several reported mechanisms, including alterations to dihydrofolatereductase (DHFR) and decreased membrane transport (36). If the ability toexert continued selection pressure is desired, then a DHFR-deficient CHOcell is recommended, along with a selection medium lacking glycine,hypoxanthine, and thymidine (37). The present hIR expression levels inthese cells are unknown. However, the CHO-hIR cell line is responsive toboth insulin and insulin-like growth factor 1 (IGF-1) in the low nanomolarrange, consistent with literature reports (38).

3.3. AKT1-EGFP expression vector – NIF2020The 7.2 kb plasmid pCORON1000 AKT1-EGFP, contains a bacterialampicillin resistance gene and a mammalian neomycin resistance gene (Fig3.1.). The sequence of the construct is available on CD from AmershamBiosciences on request. A detailed restriction map is shown in chapter 11,appendix A.


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3.4. Materials and equipment requiredThe following materials and equipment are required, but not provided.● Microplates. For imaging using the IN Cell Analyzer 3000,

Packard Black 96 Well ViewPlates (Packard Cat # 6005182) should beused. For assays in 384 well format, please [email protected] for recommendations.

● A CASY 1 Cell Counter and Analyzer System (Model TT) (Schärfe SystemGmbH) is recommended to ensure accurate cell counting prior to seeding.Alternatively a hemocytometer may be used.

● Environmentally controlled incubator (5% CO2, 95% relative humidity, 37 ºC)

● Imager/microscope (e.g. IN Cell Analyzer 3000)● Laminar flow cell culture bench● Tissue culture flasks (T-flasks) and pipettes● Controlled freezing rate device providing a controlled freezing rate of

1 ºC per min● Standard tissue culture reagents and facilities (see also section 5.1.1.)

3.5. IN Cell Analysis SystemThe AKT1-EGFP assay has been developed and optimized for analysis usingthe IN Cell Analyzer 3000 in conjunction with the Plasma Membrane Spotanalysis module. Please refer to the instrument user manual for details oninstrument set-up and the module manual for details on the algorithmsettings. The assay may also be imaged with the IN Cell Analyzer 1000System. For further information on either of these products, please contactAmersham Biosciences.

3.5.1. IN Cell Analyzer 3000 The IN Cell Analyzer 3000 is a line-scanning, laser-based, confocal imagingsystem, with three high-speed CCD cameras. It has been developedspecifically for performing information-rich cellular assays very rapidly and at

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7625 bp

Neomycin resistance gene

Ampicillin resistance gene

AKT1-EGFP

CMV enhancer

Chimeric intron

Synthetic poly A

SV40 late polyA

CMV promoter

SV40 enhancer/early promoter

T7 promoter

f1 ori

MluI (1103)

SalI (2554)

XhoI (1092)

BamHI (2563)

BamHI (5539)

BssHII (2093)BssHII (5142)

PvuI (665)

PvuI (3597)

PvuI (6352)

PstI (839)

PstI (1348)

PstI (1720)

PstI (4798)

NcoI (514)

NcoI (2584)

NcoI (4162)

NcoI (4458)

NcoI (5177)

pCORON1000 AKT1-EGFP

Fig 3.1.: Vector map of the supplied AKT1-EGFP expression vector


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high resolution, enabling high-throughput and high-content testing of drugcompounds.

3.5.2. Plasma Membrane Spot analysis module The Plasma Membrane Spot analysis module enables the quantification ofAKT1-EGFP translocation. It determines the level of GFP signal (AKT1expression) throughout the cell and then identifies and quantifies any brightobjects (in this case, ruffles) at the edge of the cell.

3.5.3. IN Cell Analyzer 1000The IN Cell Analyzer 1000 is a bench top automated microscope systemdesigned for imaging sub-cellular end-point assays. The system’s corecomponents are a Nikon microscope, xenon lamp and high-resolution CCDcamera. Additional optional modules include liquid handling andtemperature control to enable imaging live-cell assays over extended periodsand in real-time.

The IN Cell Analyzer 1000 has a number of complementary analysismodules as well as the capability to export images and data into othercommercial analysis packages. Currently there is no analysis module suitablefor AKT1-EGFP assay. However, a continual program of new analysis moduledevelopment is in place.

3.6. AKT1-EGFP translocation assay onepifluorescence microscopesFor speed of screening and quality of the images obtained, we recommendperforming the AKT1-EGFP assay on the IN Cell Analyzer Systems. However,it is possible to adapt the assay to be read on alternative imaging platforms.

Laboratory grade inverted epifluorescence microscopes such as the NikonDiaphot or Eclipse models or the Zeiss Axiovert model are suitable for imageacquisition. A high-quality objective (Plan/Fluor 40× 1.3 NA or similar) andepifluoresence filter sets compatible with GFP and the desired nuclear dyewill be required. A motorized stage with multi-well plate holder and a heatedstage enclosure are also recommended for assays performed onepifluorescence microscopes, and a suitable software package will berequired for image analysis.

3.7. Software requirementsIN Cell Analyzer 3000The Plasma Membrane Spot analysis module is available from AmershamBiosciences for automated image analysis of the AKT1-EGFP assay. Analyzeddata are exported as numerical files in ASCII format. ASCII format data canbe imported into Microsoft™ Excel, Microsoft Access, or any similar packagefor further data analysis as desired.

Confocal or epifluorescence microscope

Suitable software will be required for analysis of images acquired on

microscopes other than the IN Cell Analysis Systems.

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Chapter 4. Safety warnings,handling and precautions

4.1. Safety warnings Warning: For research use only. Not recommended or intended for diagnosisof disease in humans or animals. Do not use internally or externally inhumans or animals.

Handle as a potentially biohazardous material.

CAUTION! Contains genetically modified material

Genetically modified cells supplied in this package are for use in a suitablyequipped laboratory environment. Users within the jurisdiction of theEuropean Union are bound by the provisions of European Directive 98/81/ECthat amends Directive 90/219/EEC on Contained Use of Genetically ModifiedMicro-Organisms. These requirements are translated into local law, whichMUST be followed. In the case of the UK this is the GMO (Contained Use)Regulations 2000. Information to assist users in producing their own riskassessments is provided in the Regulations (in particular sections 3.3.1 and3.3.2).

Risk assessments made under the GMO (Contained Use) Regulations 2000for our preparation and transport of these cells indicate that containment 1is necessary to control risk. This risk is classified as GM Class 1 (lowestcategory) in the United Kingdom. For handling precautions within the UnitedStates, consult the National Institute of Health’s Guidelines for ResearchInvolving Recombinant DNA Molecules.

Instructions relating to the handling, use, storage and disposal of geneticallymodified materials:

1 These components are shipped in liquid nitrogen vapor. To avoid the riskof burns, extreme care should be taken when removing the samples from thevapor and transferring to a liquid nitrogen storage unit. When removing thecells from liquid nitrogen storage and thawing there is the possibility of anincrease in pressure within the vial due to residual liquid nitrogen.Appropriate care should be taken when opening the vial.

2 Genetically modified cells supplied in this package are for use in asuitably equipped laboratory environment, and should only be used byresponsible persons in authorised areas. Care should be taken to preventingestion or contact with skin or clothing. Protective clothing, such aslaboratory overalls, safety glasses and gloves should be worn whenevergenetically modified materials are handled.

3 Avoid actions that could lead to the ingestion of these materials. NOsmoking, drinking or eating should be allowed in areas where geneticallymodified materials are used.


● 2

4. Any spills of genetically modified material should be cleaned immediatelywith a suitable disinfectant.

5. Hands should be washed after using genetically modified materials.

6. Care should be taken to ensure that the cells are NOT warmed if they areNOT being used immediately. To maintain viability DO NOT centrifuge thecells upon thawing.

7. Most countries have legislation governing the handling, use, storage,disposal and transportation of genetically modified materials. Theinstructions set out above complement Local Regulations or Codes ofPractice and users of these products MUST make themselves aware of andobserve the Local Regulations or Codes of Practice, which relate to suchmatters.

For further information, refer to the material safety data sheet(s) and / orsafety statement(s).

4.2. StorageThe AKT1-1EGFP expression DNA construct (NIF2020) should be stored at -15 ºC to -30 ºC. The CHO derived cells expressing the AKT1-1EGFP fusion protein (NIF2019)should be stored at -196 ºC in liquid Nitrogen.

4.3. Handling

4.3.1. VectorUpon receipt, the vector should be removed from the cryo-porter and storedat -15 ºC to -30 ºC until used.After thawing the DNA sample, centrifuge briefly to recover the contents.

4.3.2. CellsThe cells should be removed from the cryo-porter and transferred to agaseous phase liquid Nitrogen storage unit. Care should be taken to ensurethe the cells are not warmed unless they are required immediately.Do not centrifuge the cell samples upon thawing.

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Chapter 5. Cell assay design

5.1. Culture and maintenance of CHO derived AKT1-EGFP expressing cell line

5.1.1. Tissue culture media and reagents required The following media and buffers are required to culture, maintain andprepare the cells, and to perform the assay.

● GIBCO™ Nutrient Mixture F-12 Ham medium with Glutamax, Invitrogen™life technologies 31765-027 or equivalent● Fetal Bovine Serum (FBS), JRH Biosciences, 12103 or equivalent. ● GIBCO Penicillin-Streptomycin (P/S), (5000 units/ml Penicillin G Sodiumand 5000 µg/ml Streptomycin Sulfate), Invitrogen life technologies 15070-063 or equivalent● Geneticin (G418), Sigma G-7034 or equivalent● GIBCO Trypsin-EDTA in HBSS w/o Calcium or Magnesium, Invitrogen lifetechnologies 25300-054 or equivalent● GIBCO Phosphate-Buffered Saline (PBS) Dulbecco’s, w/o Calcium,Magnesium or Sodium Bicarbonate, Invitrogen life technologies 14190-094or equivalent● Dimethylsulfoxide (DMSO), Sigma D-2650 or equivalent● Insulin-like Growth Factor 1 (IGF-1), human recombinant expressed in E.coli, Sigma I-3769 or equivalent● Insulin, Sigma I-0259 or equivalent● Wortmannin, Sigma W-1628 or equivalent● Acetic acid, glacial, AnalaR, BDH, 10001AS● Hoechst™ 33342, Molecular Probes H-21492 or similar● DRAQ5™, Biostatus● Bovine Serum Albumin (BSA), Sigma A-7888 or equivalent● GIBCO HEPES Buffer Solution, 1 M, Invitrogen life technologies 15630-056 or equivalent● Cy5™ monocarboxyl dye, Amersham Biosciences PA05111● Alexa Fluor 350, carboxylic acid, succinimidyl ester, Molecular Probes A-10168● Oregon Green 488, 2’, 7’-difluorofluorescein, Molecular Probes D-6145● Formalin solution (10%), neutral-buffered, 4% (w/v) Formaldehyde, SigmaHT50-1-2● Phosphate Buffered Saline tablets, Sigma, P-4417● Standard tissue culture plastic-ware including tissue culture treated flasks (T-flasks), centrifuge tubes and cryo-vials

5.1.2. Reagent preparation NOTE : the following reagents are required, but not supplied

● Heat inactivated FBS: Heat for 30 minutes in a water bath at 56 ºC● Growth-medium: Nutrient Mixture F-12 Ham medium with Glutamax supplemented with 10% (v/v) FBS, 1% (v/v) Penicillin-Streptomycin, and 0.5 mg/ml Geneticin● Freeze-medium: Nutrient Mixture F-12 Ham medium with Glutamax


● 2

supplemented with 10% (v/v) FBS, 1% (v/v) Penicillin-Streptomycin, and10% (v/v) DMSO

● Assay-medium: Nutrient Mixture F-12 Ham medium with Glutamaxsupplemented with 10 mM HEPES, 0.1% (v/v) BSA, and 1.0 µM Hoechst● 100 mM Acetic acid solution in distilled water● 50 µg/ml IGF-1: IGF-1 reconstituted as recommended by the supplier. Add0.5 ml of 100 mM acetic acid in distilled water to 50 µg of IGF-1. Add 0.5 ml of PBS to further dilute. The stock solution of 50 µg/ml IGF-1, 50 mM Acetic acid can be aliquoted and stored between -20 ºC and -70ºCuntil required. On the day of the assay the stock solution of IGF-1 is furtherdiluted in Assay-medium for use in the assay (see sections 5.2.4. and5.2.6. for further details).● Wortmannin: Wortmannin is light sensitive. Care must be taken in handlingto prevent excessive degradation. Add 5 mg Wortmannin to 0.5 ml DMSO.Wortmannin is stable in DMSO at 4 °C for 2–3 weeks only. On the day of the assay, make up to 100 ml using PBS, to give a working stock of 117 µM. This should be kept in the dark at 4 ºC throughout the assay whenever possible. Prepare a 1500 nM working dilution with Assay-medium(three fold of the final concentration, see section 5.2.6. for further details) less than an hour before it is required on the day of the assay, keeping this solution in the dark whenever practical. If a large number of assays are being performed over time during a day, we recommend preparing fresh working dilutions at regular intervals, with no working solution used once it is 2 h old.● Flat field (FF) solution components:

● Cy5 - 1 mM stock solution prepared in 10% (v/v) DMSO, 90% (v/v) PBS● Alexa Fluor 350 - 1 mM stock solution prepared in 10% (v/v) DMSO,90% (v/v) PBS● Oregon Green 488 - 1 mM stock solution prepared in 10% (v/v) DMSO,90% (v/v) PBS

As explained in the IN Cell Analyzer 3000 user manual, prepare the FFsolution to give adequate fluorescent signal in each channel used, where thefluorescent counts should be less than 3300, at maximum.For a Hoechst 33342 nuclear stained assay, prepare an initial FF solutioncontaining 4.5 µl 10 µM Oregon Green 488 and 20 µl 100 µM Alexa Fluor350 in 100 µl PBS.For a DRAQ5 nuclear stained assay, use 4.5 µl 10 µM Oregon Green 488and 10 µl 10 µM Cy5 in 100 µl PBS.Adjust these solutions if required. Use 100 µl of FF solution for a 96 wellplate and 40 µl FF solution for a 384 well plate.

5.1.3. Cell thawing procedure Two cryo-vials, each containing 1 x 106 cells in 1 ml of Freeze-medium areincluded with this assay kit. The vials are stored frozen in the vapor phase ofliquid Nitrogen.1 Remove a cryo-vial from storage.2 Holding the cryo-vial, dip the bottom three-quarters of the cryo-vial into a37 ºC water bath, and swirl gently for 1–2 min until the contents arethawed. Do not thaw the cells for longer than 3 min as this decreasesviability.

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3. Remove the cryo-vial from the water bath and wipe it with 70% (v/v)ethanol. Transfer the cells immediately to a T-25 flask and add 5 ml pre-warmed Growth-medium drop-wise to prevent cell damage. Add a further2ml Growth-medium and incubate at 37 ºC.NOTE: To ensure maximum cell viability, do not allow the cells to thaw atroom temperature and do not thaw the cells by hand.

5.1.4. Cell sub-culturing procedure Incubation: 5% CO2, 95% humidity, 37 ºC.The cells should be passaged at ratio of 1:10 when they are 70% confluent.1 Warm all reagents to 37 ºC.2 Aspirate the medium from the cells and discard.3 Wash the cells with PBS. Take care not to damage the cell layer whilewashing, but ensure that the entire cell surface is washed.4 Aspirate the PBS from the cells and discard.5 Add Trypsin-EDTA (2 ml for T-75 flasks and 4ml for T-162 flasks),ensuring that all cells are in contact with the solution. Wait for 1–3 min forthe cells to round up / loosen. Check on an inverted microscope.6 When the cells are loose, tap the flask gently to dislodge the cells. AddGrowth-medium (8 ml for T-75 and 6 ml for T-162 flasks) and gentlyresuspend the cells with a 10 ml pipette until all the clumps have dispersed.7 Aspirate the cell suspension and dispense 1 ml cells into a new culturevessel.

5.1.5. Cell seeding procedure The following procedure is optimized for cells grown in standard T-75 and T-162 flasks to be seeded into 96 well microplates.1 Warm all reagents to 37 ºC.2 Aspirate the medium from the cells and discard.3 Wash the cells with PBS. Take care not to damage the cell layer whilewashing, but ensure that the entire cell surface is washed.4 Aspirate the PBS from the cells and discard.5 Add Trypsin-EDTA (2 ml for T-75 and 4 ml for T-162 flasks), ensuring thatall cells are in contact with the solution.Immediately aspirate Trypsin-EDTAfrom the cells. Wait for 1–3 min for the cells to round up / loosen. Check onan inverted microscope.6 When the cells are loose, tap the flask gently to dislodge the cells. AddGrowth-medium (10 ml) and gently resuspend the cells with a 10 ml pipetteuntil all the clumps have dispersed.7 Centrifuge the cell suspension at 1400 rpm for 3 min. Discard thesupernatent and resuspend the pellet of cells with 4 ml Growth-medium.8 Count the cells using either a CASY1 Cell Counter and Analyzer System(Model TT) or a hemocytometer.9 Using fresh Growth-medium, adjust the cell density to deliver the desirednumber of cells to each well. For example, to add 0.6 x 104 cells per well ina volume of 200 µl, adjust the suspension to 3 x 104 cells per ml. Werecommend a concentration of 3–5 x 104 cells per ml.10 Dispense 200 µl of the cells into each well of the microplate, except thewell reserved for the FF solution (see IN Cell Analyzer 3000 manual forfurther information).


● 4

11 Optionally incubate the plates undisturbed on a level surface for 1 h atroom temperature (approximately 20 ºC). This treatment may reduce edgeeffects.12 Incubate the plated cells for 24 h at 37 ºC, 5% CO2, 95% humidity beforestarting the assay.N.B. If the cells are near confluence prior to trypsinization, they should bepassaged into two T-flasks. They will then be ready for seeding the followingday

5.1.6. Cell freezing procedure 1 Harvest and centrifuge the cells as described in section 5.1.5 andresuspend the cells in a small volume of freeze-medium.2 Count the cells as described in section 5.1.5.3 Adjust the cell suspension with freeze-medium to a concentration of 1 x 106

cells per ml and transfer into cryo-vials. Each vial should contain 1 x 106 cellsin 1 ml of freeze-medium.4 Transfer the vials to a cryo-freezing device and freeze at -80 ºC for 16–24 h.5 Transfer the vials to the vapor phase in a liquid nitrogen storage device.

5.1.7. Growth characteristics Under standard growth conditions, the cells should maintain an average size of18.5 µm as measured using a CASY1 Cell Counter and Analyzer System (ModelTT). The doubling time of the cell line in exponential growth phase has beendetermined to be approximately 12.3 h under standard conditions (Fig 5.1.).

5.2. Assay set up

5.2.1. Live cell AKT1-EGFP assay using the IN Cell Analyzer 3000 This manual provides a suggested protocol to use the AKT1-EGFP assay forboth agonist and antagonist screening on the IN Cell Analyzer 3000.

5.2.2. Microplate set up for 96 well format assays It is essential that the number of cells per well in the assay plates beconsistent in order to minimize assay variability. IGF-1 is used as a reference agonist with a typical EC50 value of 23 ng/ml.Wortmannin is used as a reference antagonist with a typical IC50 value of 15.8nM. The AKT1-EGFP assay can be used with either Hoechst or DRAQ5 as theNuclear-stain.

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0 25 50 75 100 125

6

8

10

12

14

Time (h)

In (cell nu

In (

cell

num

ber)

Fig 5.1. Growth curve of CHO derivedAKT1-EGFP expressing cell line (onlypoints on the linear portion of the curveis shown). Doubling time = 12.3 h.


● 5

As explained in the IN Cell Analyzer 3000 user manual, each run mustcontain a flat field well to compensate for variations in fluorescenceintensity across each image. It is possible to prepare a plate solely for thispurpose. Alternatively, a designated well on each plate can contain flatfield solution. When seeding the plate, the flat field well must not containany cells if the auxiliary flat field correction tool is to be applied in theanalysis module.

5.2.3. Schematic agonist assay protocol Fig 5.2. Shows a typical schematic of an agonist assay. The cells should beseeded in the microplate the day before the experiment. One hour after theaddition of the Assay-medium with Nuclear stain, the test compounds areadded, and imaging occurs at the peak translocation time 4 min later. Thetime taken to image each well of an entire microplate needs to be takeninto consideration when dispensing test compounds in this live cell assay.Alternatively for convenience, the live cell assay could be fixed at the peaktranslocation time point and then imaged on the IN Cell Analyzer 3000(see section 5.2.9. for further details).

5.2.4. Agonist assay protocol (96 well format) NOTE: whenever possible, keep the microplate at 37 °C, 5% CO2, and95% humidity. 1 The day before starting the assay, seed 0.6 x 104 cells per well in 200 µl

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Fig 5.2. Flow diagram showing a basicprotocol suitable for a AKT1-EGFPagonist screen. This flowchartillustrates how a AKT1-EGFP endpointagonist screen could be performed witha 4 min peak translocation time.

Add Assay-medium with Nuclear-stain.

Incubate 1 h, 37 oC, 5% CO2.

Add compounds—timed dispense.

Incubate 4 min, 37 oC, 5% CO2.

Move plate to IN Cell Analyzer 3000.

Image plate.

Seed cells.

START

Incubate overnight, 37 oC, 5% CO2.

Decant, Wash, Decant.

STOP.


● 6

of Growth-medium. Incubate for 24 h at 37 ºC, 5% CO2. If one of the wellson the cell plate is used for FF correction, it should not contain cells.2 On the day of the assay, prepare the test compounds, solvent controls (ifused) and reference agonist controls (IGF-1). These samples are typicallyprepared at four fold of the final concentration in Assay-medium. For thereference agonist IGF-1, a final concentration of 2 µg/ml is suitable (see5.1.2 for IGF-1 reagent preparation). However, we recommend that usersperform their own dose response curve to establish optimal agonistconcentrations.3 Decant the Growth-medium from the cell plate, removing all excess liquidand add 100 µl Assay-medium to wash the cells. Decant the wash.4 Add 150 µl Assay-medium. Incubate the plates at 37 ºC, 5% CO2 for 1 h.5 Add 50 µl of the prepared four fold dilution stocks of the test and controlcompounds to the appropriate wells in a strict time-controlled manner, sothat each well has been exposed to the compound / control solutions for 4 min by the time that well is imaged. The total well volume is 200 µl.6 After the first well has been incubated for 4 min, read the assay plateusing the IN Cell Analyzer 3000. Alternatively for convenience, the live cellassay could be fixed at the peak translocation time point and then imagedon the IN Cell Analyzer 3000 (see section 5.2.9. for further details).7 Perform the data analysis using the Plasma Membrane Spot analysismodule.

5.2.5. Schematic antagonist assay protocolFig 5.3. shows a typical schematic of an antagonist assay. The cells shouldbe seeded in the microplate the day before the experiment. 1 h after theaddition of the Assay-medium with Nuclear stain and the test compounds,the agonist is added and imaging occurs occurs at the peak translocationtime 4 min later.

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Incubate overnight, 37 oC, 5% CO2.

Add Assay-medium with Nuclear-stain.

Add test and control compounds

Incubate 1 h, 37 oC, 5% CO2.

Move plate to IN Cell Analyzer 3000.

Image & dispense agonist into each well.

Image each well at 4 min.

STOP.

Seed cells.

START

Decant, Wash, Decant.

Fig 5.3. Flow diagram showing a basicprotocol suitable for an AKT1-EGFPantagonist screen. This flowchartillustrates how an AKT1-EGFP endpointantagonist screen could be performedwith a 4 min peak translocation time.

5.2.6. Antagonist assay protocol (96 well format) Note: whenever possible, keep the microplate at 37 °C, 5% CO2, and 95%humidity.1 The day before starting the assay, seed 0.6 x 104 cells per well in 200 µlof Growth-medium. Incubate for 24 h at 37ºC, 5% CO2. If one of the wellson the cell plate is used for flat field correction, it should not contain cells.2 On the day of the assay, prepare the solvent controls (if used), referenceantagonist control (Wortmannin) and test compounds. These samples aretypically prepared at three fold of the final concentration in Assay-medium.For Wortmannin, a final concentration of 500 nM is suitable (see 5.1.2 forWortmannin reagent preparation). However, due to Wortmannin instability,we recommend that users perform their own inhibition curve to establish anoptimal antagonist concentration. Also prepare a four-fold stock of thereference agonist (IGF-1) in Assay-medium. For the reference agonist IGF-1,a final concentration of 1 µg/ml is suitable (see 5.1.2 for IGF-1 reagentpreparation).3 Decant the Growth-medium from the cell plate, removing all excess liquidand add 100 µl Assay-medium to wash the cells. Decant the wash.4 Add 100 µl of Assay-medium to the wells.5 Add 50 µl of the prepared three-fold dilution stocks of the test andcontrol compounds to the appropriate wells. The total well volume is now150 µl.6 Incubate the plates at 37ºC, 5% CO2 for 1 h.7 Read the assay plate before agonist addition, using the IN Cell Analyzer3000. This is the T0 read.


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8 Immediately after each well is read, use the IN Cell Analyzer 3000 to add50 µl of the four-fold reference agonist solution to that well. Therecommended final concentration of IGF-1 is 1 µg/ml. The total well volumeis now 200 µl.9 After the first well has been incubated for 4 min, read the assay plateagain, using the IN Cell Analyzer 3000. This is the T1 read.10 Perform data analysis using the Plasma Membrane Spot Analysis module.

5.2.7. Timing schedule Since the maximal translocation of AKT1-EGFP occurs 4 min after theaddition of the agonist (IGF-1), timing is critical. Following the suppliedprotocols, scanning an entire 96 well microplate using a two pass protocol(required when using Hoechst 33342 and GFP) will take greater than 4 min.Therefore, the off-line addition of test agonists should be performed in atimed manner, either manually or automatically, to ensure that each well ofthe plate is incubated for the optimal time.

DRAQ5 stained cells can be imaged more quickly than Hoechst 33342stained cells because the GFP and Hoechst signals must be imagedsequentially due to overlap of the spectral profiles of these two probes.DRAQ5 and GFP can be imaged simultaneously (see Fig 5.15. for furtherinformation).

An alternative approach, which avoids the necessity of timed dispensing, isto configure the assay in a fixed cell format (see section 5.2.9. for furtherdetails).

Another approach is to increase the length of time of agonist stimulation togreater than 4 min. This may decrease the magnitude of response (seesection 5.6.6), but may enable the entire 96 well plate to be imaged in onerun.

5.2.8. Important considerationsWhen performing an antagonist screen, it is important to remember that thetest compound added to the plates will be diluted by addition of the agonist.It is recommended that the cells incubate in 1 x target test compoundsolution prior to the addition of the agonist. Upon addition of agonist, thetest compound concentration will therefore be 75% of the target testcompound concentration. Other options are available and can be determinedby the user.

5.2.9. Fixed cell assay formatIn order to avoid the need for strict time-controlled dispensing, it is possibleto complete the assay using live cells, as described in the agonist andantagonist assay protocols, but then fix the cells prior to imaging andanalysis.1. Perform the assay as described.2. After incubating with agonist for the optimal time, decant the Assay-medium and wash the cells in each well with 200 µl PBS.3. Add 100 µl 2% (w/v) Formaldehyde in PBS to each well and incubate for


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30 min at room temperature.4. Decant the fixative and wash the cells in each well with 200 µl PBS.5. Store the plate at 4 ºC with 100 µl fresh PBS in each well.6. Image and perform analysis as required.

If the cells are to be fixed, the assay can be performed with or without theNuclear-stain in the Assay-medium for the duration of the assay. Nuclearstaining, at the same concentrations as normal, can be performed afterfixation. If no stain is used during the assay, the first PBS wash step can beomitted

5.3. Results

5.3.1. Calculating the Z’-factorAssay performance can be assessed by calculating the Z’-factor, adimensionless value defined by Zhang et al. (39). Using the IN Cell Analyzer3000, a Z’-factor of >0.3 should be obtained with the assay under standardconditions, if the experiment is performed as described in this manual.

where σ = standard deviationµ = mean signalc+ = positive controlc– = negative control

5.3.2. Example resultsThe data shown in figures 5.4 and 5.5 were generated from a singleexperiment, and provide an example of the images and results that areobtainable with the AKT1-EGFP assay using the IN Cell Analyzer 3000 andPlasma Membrane Spot Analysis Module. Fig 5.4. shows images taken onthe IN Cell Analyzer 3000 of the supplied AKT1-EGFP expressing cellsbefore and after stimulation with 2 µg/ml IGF-1

Images shown are without nuclear staining and are 1/20th of the actualimage size acquired by the IN Cell Analyzer 3000.

−cc

c )33(1'

µµσσc

−+−=

+

−+Z

Fig 5.4. The same group of cellsexpressing AKT1-EGFP before (A) andafter (B) stimulation with 2 µg/ml IGF-1.


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Results indicate a Z’-factor of 0.60 for stimulated versus unstimulatedcells.

Fig 5.5. Data from the exampleexperiment, generated by the PlasmaMembrane Spot analysis module,exported to and analyzed in MicrosoftExcel.


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5.4. Assay characterization

5.4.1. Translocation indexAll validation assays for the AKT1-EGFP assay were performed on the INCell Analyzer 3000 using the Plasma Membrane Spot analysis module. Thedata generated by this module is in the format of Aspots. Aspots are definedas the total area of identified spots (ruffles) per cell, averaged for allidentified cells in a well. This translocation index is used in all the followingdata.

5.4.2. Summary of quantitative assay parametersA summary of typical assay data, using IGF-1 as the agonist, is shown inTables 5.1. and 5.2. In particular, Table 5.1. shows the results obtainedfrom a single assay plate, indicating the level of well to well variation. Table5.2. shows a summary of the results obtained from 19 assays, performed bydifferent operators on different occasions, giving an indication of inter assayvariation.

Parameter Assay Data # Assays # ReplicatesSignal to Noise 30.65 1 18Z'-factor 0.56 1 18Magnitude of Response 116.28 1 18%CVStimulated 9.99 1 18Unstimulated 25.78 1 18

Parameter Assay Data (± SD*)# Assays # ReplicatesSignal to Noise 27.20 ± 12.68 19 18Z'-factor 0.48 ± 0.14 19 18Magnitude of Response 115.39 ± 11.03 19 18%CVStimulated 10.92 ± 2.51 19 18Unstimulated 27.12 ± 5.63 19 18

Table 5.1. Results from a typical singleassay, performed using the suggestedprotocolSignal to noise is (mean signal – meanbackground)/(background standarddeviation) (39)Magnitude of response is (mean signal– mean background)% CV is (standard deviation x100/mean)Z’-factor is a dimensionlesscharacteristic useful for evaluation ofassay quality (39)

Table 5.2. Summary results from assaysperformed by different operators ondifferent occasions, using the suggestedprotocol* SD shown is the standard deviation ofthe assaysSignal to noise is (mean signal – meanbackground)/(background standarddeviation) (39)Magnitude of response is (mean signal -mean background) % CV is (standard deviation x 100/mean)Z’-factor is a dimensionlesscharacteristic useful for evaluation ofassay quality (39).


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2000 4000 6000 8000 100000

50

100

150 Control (no IGF-1)

2 µg/ml IGF-1

Cell Density (cells / well)

Aspots

Tran

sloc

atio

n In

dex

(Asp

ots)

Fig 5.6. IGF-1-induced AKT1-EGFPtranslocation as a function of seedingdensity. Samples were prepared in a96-well microplate. Stimulated cellswere treated with 2 µg/ml IGF-1 for 4min 45 s prior to imaging. Error = ± SD, n = 4 replicates per datapoint.

5.4.3. Seeding densityFig 5.6. shows the relationship of seeding density to assay response. Thedata were collected approximately 4 min 45 s after the addition of 2 µg/ml IGF-1. Significant differences between stimulated (2 µg/ml IGF-1)and non stimulated cells (Control – no IGF-1) were detected at celldensities ranging from 0.2 x 104 to 1 x 104 cells per well. We recommendseeding the cells at a density of 0.6 x 104 cells per well.

5.4.4. IGF-1 dose responseFig 5.7. shows an agonist dose response curve for the supplied cells to IGF-1. The data were collected 4 min after addition of the agonist, anddemonstrate an EC50 of 23 ng/ml.


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-10 -9 -8 -7 -6 -50

50

100

150

log IGF-1 (g/ml)

Aspots

Asp

ots

log [IGF-1] (g/ml)

Tran

sloc

atio

n In

dex

(Asp

ots)

Fig 5.7. IGF-1 dose response curveusing the supplied AKT1-EGFP cellline. Error = ± SD, n = 8 replicates per datapoint.

5.4.5. Wortmannin inhibition curve

Fig 5.8. shows a typical inhibition curve for Wortmannin. Data werecollected approximately 4 min 45 s after the addition of 1 µg/ml IGF-1 tocells with varying concentrations of Wortmannin. An IC50 of 15.8 nM wasdetermined.

-11 -10 -9 -8 -7 -6 -5

0

50

100

150

log Wortmannin (M)

Aspots

Asp

ots

Tran

sloc

atio

n In

dex

(Asp

ots)

log [Wortmannin] (M)

Fig 5.8. Wortmannin inhibition curve, inthe presence of 1 µg/ml IGF-1, usingthe supplied AKT1-EGFP cell line.Error = ± SD, n = 4 replicates per datapoint.


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0.00% 0.25% 0.50% 1.00% 2.00%0

50

100

150

1 µg/ml IGF-1

1 µg/ml IGF-1, 500 nM wortmannin

DMSO %

Aspots

Asp

ots

DMSO %

Tran

sloc

atio

n In

dex

(Asp

ots)

Fig 5.10. The effects of DMSO, ethanolor methanol on the AKT1-EGFPtranslocation. Error = ± SD, n = 4 replicates per datapoint.

5.4.6. IGF-1 agonist time courseFig 5.9. shows a typical time course of the AKT1-EGFP translocation onstimulation with 2 µg/ml IGF-1 and indicates that the maximaltranslocation occurs 4–6 min after the addition of 2 µg/ml IGF-1.

5.4.7. Sensitivity of assay to DMSO, ethanol and methanol The AKT1-EGFP translocation was measured in the presence of DMSO (≤ 2%), Ethanol (≤ 2%) or Methanol (≤ 2%). Fig 5.10. shows the AKT1-EGFP translocation 4 min after stimulation with either 1 µg/ml IGF-1 onlyor 1 µg/ml IGF-1 plus, 500 nM Wortmannin in the presence of varying finalconcentrations of DMSO, Ethanol or Methanol. As can be seen in Fig 5.10.the assay can tolerate up to 0.25% DMSO, up to 0.50% Ethanol or up to2.00% Methanol.

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 190

50

100

150

Control (no IGF-1)

2 µg/ml IGF-1

Time (min)

Aspots

Asp

ots

Time (min)

Tran

sloc

atio

n In

dex

(Asp

ots)

Fig 5.9. Time course of AKT1-EGFPtranslocation using 2 µg/ml IGF-1 as anagonist. Maximal response is seen after4–6 min. Error = ± SD, n = 3 replicates per IGF-1 data point and n = 2 replicates percontrol data point.


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0.00% 0.25% 0.50% 1.00% 2.00%0

50

100

150

1 µg/ml IGF-1

1 µg/ml IGF-1, 500 nM wortmannin

Ethanol %

Aspots

Asp

ots

Ethanol %Tr

ansl

ocat

ion

Inde

x(A

spot

s)

0.00% 0.25% 0.50% 1.00% 2.00%0

50

100

150

1 µg/ml IGF-1

1 ug/ml IGF-1, 500 nM wortmannin

Methanol %

Aspots

Asp

ots

Methanol %

Tran

sloc

atio

n In

dex

(Asp

ots)

5.4.8. Effects of different Assay-mediumTo determine the effects of a variety of assay-media on the IGF-1 inducedAKT1-EGFP translocation, cells were assayed in 10 different formulations ofAssay-medium. The data were acquired approximately 4 min 35 s after theaddition of control (no IGF-1) or 2 µg/ml IGF-1. The results, shown in Fig 5.11, demonstrate that the assay can tolerate a range of different mediabut is optimal in the recommended Assay-medium (Nutrient Mixture F-12Ham medium with Glutamax supplemented with 10 mM HEPES, 0.1 %(v/v) BSA, and 1.0 µM Hoechst).


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5.4.9. Effects of serum starvationTo determine the effect of the duration of serum starvation of cells on theAKT1-EGFP translocation, cells were incubated in assay media for 0 - 4 hprior to the assay. The results, shown in Fig 5.12, show that a 1 h starvationperiod in the recommended Assay-medium is optimal for the assay.

Ham

s F-1

2

Ham

s F-1

2, 1

0 m

M H

EPES

Ham

s F-1

2, 1

0 m

M H

EPES, 0

.1%

BSA

Ham

s F-1

2, 1

0 m

M H

EPES, 0

.2%

BSA

Ham

s F-1

2, 1

0 m

M H

EPES, 1

.0%

FCS

Ham

s F-1

2, 1

0 m

M H

EPES, 5

.0%

FCS

Ham

s F-1

2, 1

0 m

M H

EPES, 1

0.0%

FCS

Krebs

Ringe

r (KRW

)

Krebs

Ringe

r (KR

W),

0.1%

BSA

Krebs

Ringe

r (KR

W),

0.2%

BSA

0

50

100

150

Control (no IGF-1)

2 µg/ml IGF-1

Assay-medium formulation

Tra

nslo

cati

on

In

dex

(Asp

ot) s

Fig 5.11. Effect of different assaymedia on IGF-1 induced AKT1-EGFPtranslocation. Error = ± SD, n = 4replicates per data point


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5.4.10. Fixed AssayIn order to avoid the need for strict time-controlled dispensing the cells canbe fixed at the endpoint of the assay prior to imaging and analysis. For fulldetails see section 5.2.9. Fig 5.13. shows AKT1-EGFP translocation assayresults after fixation with 2% Formaldehyde in PBS prior to imaging on theIN Cell Analyzer 3000.

5.4.11. Insulin agonistInsulin can be used instead of IGF-1 as an agonist. Fig 5.14. shows atypical time course of AKT1-EGFP translocation after stimulation with 1 µMInsulin. Maximal translocation occurs 3–4 min after the addition of Insulin.

0 1 2 3 4

0

50

100

150

Control (no IGF-1)

2 µg/ml IGF-1

Starvation time (h)

Aspots

Asp

ots

Tran

sloc

atio

n In

dex

(Asp

ots)

Starvation time (h)

Fig 5.12. Effect of serum starvation onIGF-1 induced translocation of AKT1-EGFP. Error = ± SD, n = 4 replicates per datapoint.

Control (no IGF-1) 1 µg/ml IGF-10

50

100

150

Aspots

Asp

ots

Tran

sloc

atio

n In

dex

(Asp

ots)

Fig 5.13. IGF-1-induced AKT1-EGFPtranslocation. Cells were incubated inthe absence or presence of (Control)1 µg/ml IGF-1 for 4 min prior to fixationand imaging. Error = ± SD, n = 48replicates per data point.


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5.4.12. DRAQ5 nuclear stainFig 5.15. shows an inhibition curve for Wortmannin using 1 µM DRAQ5Nuclear stain in place of Hoechst in the Assay-medium. Data were collectedapproximately 4 min 28 s after the addition of 1 µg/ml IGF-1 and varyingconcentrations of Wortmannin. An IC50 of 6.0 nM was determined. The datademonstrate that DRAQ5 is a suitable alternative nuclear stain to Hoechstfor use in the AKT1-EGFP assay.

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 190

50

100

150

Control (no Insulin)

1 µM Insulin

Time (min)

Aspots

Time (min)Tr

ansl

ocat

ion

Inde

x(A

spot

s)

Fig 5.14. Time course of AKT1-EGFPtranslocation in response to 1 µMInsulin treatment. Maximal response is seen after 3–4min.Error = ± SD, n = 4 replicates perInsulin data point and n = 1 replicateper control data point.

-11 -10 -9 -8 -7 -6 -50

50

100

150

log [Wortmannin] (M)

Aspots

Tran

sloc

atio

n In

dex

(Asp

ots)

Fig 5.15. Wortmannin inhibition curve,in the presence of 1 µg/ml IGF-1. 1 µM DRAQ5 was used as the Nuclearstain in the Assay-medium. Error = ± SD, n = 4 replicates per datapoint.


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5.4.13. Results obtained from the IN Cell Analyzer 1000Fig 5.16 shows typical images of AKT1-EGFP translocation obtained usingthe IN Cell Analyzer 1000. Assays were set up as described in section 5.2.

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Fig 5.16. CHO-hIR cells expressingAKT1-EGFP treated for 4 min withassay buffer only (A); agonist only - 1µg/ml IGF-1 (B); or agonist in thepresence of antagonist - 1 µg/ml IGF-1in presence of 500 nM Wortmannin(C).The assay was performed aspreviously detailed. Cells were fixedwith 2% Formaldehyde prior to imagingon IN Cell Analyzer 1000. Only EGFP-AKT1 expression shown. Images shownare only a fraction of those obtained onthe IN Cell Analyzer 1000.


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Chapter 6. Vector use detailsThe plasmid vector pCORON1000 AKT1-EGFP (Fig 3.1.) can be used totransiently or stably express the AKT1-EGFP fusion protein in the cell line ofchoice.

6.1. General guidelines for vector usepCORON1000 AKT1 EGFP has been used successfully to express AKT1-EGFP fusion protein both transiently and stably in the CHO derived cell line.Expression levels, translocation responses and other assay parameters mayvary depending on the cell type and the transfection procedure.

6.2. Transient transfection with pCORON1000 AKT1-EGFPTransient transfection protocols must be optimized for the cell type ofchoice. Choice of transfection reagent and cell type will affect efficiency oftransfection. FuGENE 6 Transfection Reagent (Roche) produced successfulresults when transfecting pCORON1000 AKT1-EGFP into CHO-hIR cells. For more information, refer to manufacturer's guidelines forthe desired transfection reagent.

6.3. Stable cell line generation with pCORON1000 AKT1-EGFP The process of establishing stable cell lines involves a large number ofvariables, many of which are cell-line dependent. Standard methods andguidelines for the generation of stable cell lines are available in the publicdomain (40).

pCORON1000 AKT1-EGFP has been used to generate stably transfected cellpopulations. The magnitude of the response and the kinetics of thetranslocation event achievable with different cell lines are unknown, and maydeviate from the values specified in this manual.


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Table 7.1.: Quality control information forAKT1-EGFP cell line

Table 7.2.: Quality control information forthe AKT1-EGFP expression vector

Table 7.3.: Expected restriction pattern forthe AKT1-EGFP expression vector

● 1

Chapter 7. Quality control

7.1. AKT1-EGFP cell line The AKT1-EGFP cell line is supplied at a concentration of 1 x 106 cells per ml in fetal calf serum containing 10% (v/v) DMSO. Thecell line has the characteristics detailed in Table 7.1.

Property Value Measurement method

Assay stability Magnitude of response Quality Control Assay ≥ 100 for 20 passages

after dispatch, Z' factor ≥ 0.3

Viability from frozen > 80 % CASY1 Cell Counter and Analyzer System (Model TT)

Cell diameter (mm) 17–20 CASY1 Cell Counter and Analyzer System (Model TT)

Fluorescence at > 20 000 for 20 FARCyte (Gain 55) 3 x 104 cells per ml passages after

(RFU) dispatch

7.2. AKT1-EGFP domain expression vector The AKT1-EGFP expression vector is supplied in TE buffer (10 mM Tris, 1 mM EDTA, pH 8.9) at 250 µg/ml. The vector has thecharacteristics outlined in Table 7.2.

Property Value Limits Measurement method

Concentration 250 µg/ml UV Absorbance @ 260 nm in water

Purity - Minimal A260/A280 ratio Between UV/Viscontamination of the 1.8–2.2 Absorbance @DNA construct by RNA 260 nm and or protein 280 nm

Expected restriction Restriction Agarose gel pattern digests should yield electrophoresis

fragments of thesizes shown in

Table 7.3.

Enzyme(s) # of cuts Fragment(s) size (bp)

NcoI 5 296, 719, 1578, 2070, 2962PvuII 3 624, 2932, 4069BamHI 2 2976, 4649PstI 4 372, 509, 3078, 3666


Problem

❶Low assay response. (positive vs.negative controls)

❷Low nuclear intensity.

❸Image is out of focus.

❹Cells do not adhere to well bottom inplate.

❺Shading across image field.

Chapter 8. Troubleshooting guide 8.1 Troubleshooting guide Possible causes and remedies

Possible cause1.1. Passage number too high.1.2. Cell density too low or too high. 1.3. Incorrect selection of analysis parameters. 1.4. Incorrect assay/incubation conditions. 1.5. Reagents were not stored properly or they are out of date. Remedy1.1. Start a fresh batch of cells from an earlier passage number. Cells shouldbe expanded, and additional vials should be frozen down from the vialsdelivered with the kit. 1.2. Verify density of cell plating; adjust plating density to values that yieldoptimal assay response. 1.3. Check that the primary parameters are correct and suitable for the cellscurrently in use. 1.4. Ensure that proper incubation is maintained as consistently as possibleduring the assay. When plates are out of the CO2 incubator for extendedperiods, it is essential that HEPES buffer is added to the medium to maintainproper pH.1.5. Repeat assay with fresh reagents.

Possible cause2.1. Nuclear stain concentration too low.2.2. Nuclear stain incubation time too short. Remedy2.1. Adjust Nuclear stain concentration to recommended level.2.2. Adjust Nuclear stain incubation time to recommended length.

Possible cause3.1. Autofocus Offset is chosen incorrectly or the system may need to berealigned.Remedy3.1. Alignment and calibration of instrument. Perform Z-stack on cells.Change Autofocus Offset.

Possible cause4.1. Plating density too high.Remedy4.1. Reduce plating density.

Possible cause5.1. flat field correction not applied or flat field solution too weak.Remedy5.1. Apply flat field correction or adjust flat field solution.

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Chapter 9. References

9.1. References1. Downward, J. Mechanisms and Consequences of Activation of ProteinKinase B/Akt. Curr Opin Cell Biol, 10, 262-267 (1998).

2. Datta, S. R. et al. Cellular Survival: a Play in Three Akts. Genes Dev, 13,2905-2927 (1999).

3. Hill, M.M. and Hemmings, B.A. Inhibition of protein kinase B/Akt,implications for cancer therapy. Pharmacol. Ther., 93, 243-251 (2002).

4. Sun, M. et al. Akt1/PKBa kinase is frequently elevated in humancarcinomas and its constitutive activation is required for oncogenictransformation of NIH3T3 cells. Am. J. Pathol., 159, 431-437 (2001).

5. Perez-Tenorio, G. and Stal, O. Activation of AKT/PKB in breast cancerpredicts a worse outcome among endocrine treated patients. Br J Cancer.,86(4), 540-545 (2002).

6. Stal, O. et al. Akt kinases in breast cancer and the results of adjuvanttherapy. Breast Cancer Res., 5, R37-R44 (2003).

7. Staal, S.P. Molecular cloning of the akt oncogene and its humanhomologues AKT1 and AKT2: amplification of AKT1 in a primary humangastric adenocarcinoma. Proc. Natl. Acad, Sci USA, 84, 5034-5037 (1987).

8. Izuishi, K. et al. Remarkable tolerance of tumor cells to nutrientdeprivation: possible new biochemical target for cancer therapy. Cancer Res.60(21), 6201-6207 (2000).

9. Andjelkovic, M. et al. Role of Translocation in the Activation and Functionof Protein Kinase B. J Biol Chem, 272, 31515-31524 (1997).

10. Alessi, D. R. et al. 3-phosphoinositide-dependent protein kinase-1 (PDK-1): Structural and functional homology with the Drosophila DSTPK61 kinase.Curr Biol, 7, 776-789 (1997).

11. Alessi, D. R. et al. Characterization of a 3-phosphoinositide-dependentprotein kinase which phosphorylates and activates protein kinase B. CurrBiol, 7, 261-269 (1997).

12. Currie, R. A. et al. Role of phosphatidylinositol 3,4,5-trisphosphate inregulating the activity and localization for 3-phosphoinositide-dependentprotein kinase-1. Biochem J, 337, 575-583 (1999).

13. Alessi, D. R. et al. Molecular basis for the substrate specificity of proteinkinase B, comparison with MAPKAP kinase-1 and p70 S6 kinase. FEBS Lett,399, 333-338 (1999).

14. Walker, K. S. et al. Activation of protein kinase B beta and gammaisoforms by insulin in vivo and by 3-phosphoinositide-dependent proteinkinase-1 in vitro: Comparison with protein kinase B alpha. Biochem J, 331,229-308 (1998).

15. Reed, J. C. Bcl-2 family proteins. Oncogene, 17, 3225-3236 (1998).

16. Muslin, A. J. et al. Interaction of 14-3-3 with signalling proteins ismediated by the recognition of phosphoserine. Cell, 84, 889-897 (1996).

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17. Yaffe, M. B. et al. The structural basis for 14-3-3: Phosphopeptide bindingspecificity. Cell, 91, 961-971 (1997).

18. Cardone, M. H. et al. Regulation of cell death protease caspase-9 byphosphorylation. Science, 282, 1318-1321 (1998).

19. Biggs, W. H. et al. Protein kinase B/Akt-mediated phosphorylation promotesnuclear exclusion of the winged helix transcription factor FKHR1. Proc NatlAcad Sci, 96, 7421-7426 (1999).

20. Brunet, A. et al. Akt promotes cell survival by phosphorylating andinhibiting a Forkhead transcription factor. Cell, 96, 857-868 (1999).

21. Mercurio, F. and Manning, A. M. Multiple signals converging on NFkB. CurrBiol, 11, 226-232 (1999).

22. Kane, L. P. et al. Induction of NFkB by the Akt/PKB kinase. Curr Biol, 9,601-604 (1999).

23. Rossig, L. et al. Glycogen Synthase Kinase-3 Couples AKT-DependentSignaling to the Regulation of P21Cip1 Degradation. J Biol Chem, 277, 9684-9689 (2002)

24. Burgering, B. M. T. and Coffer, P.J. Protein kinase B (c-akt) inphosphatidylinositol 3-kinase signalling. Nature, 376, 599-602 (1995).

25. Wang, Q. et al. Protein kinase B/Akt participates in GLUT4 translocation byinsulin in L6 myocytes. Mol Cell Biol, 19, 4008-4018 (1999).

26. Magun, R. et al. Expression of a constitutively active form of protein kinaseB (c-akt) in 3T3-L1 preadipose cells causes spontaneous differentiation.Endocrinology, 137, 3590-93 (1996).

27. Brennan, P. et al. Phosphatidylinositol 3-kinase couples the interleukin-2receptor to the cell cycle regulator E2F. Immunity, 7, 679-689 (1997).

28. Deprez, J. et al. Phosphorylation and Activation of Heart 6-Phosphofructo-2-Kinase by Protein Kinase B and Other Protein Kinases of the InsulinSignaling Cascades. J Biol Chem, 272, 17269-17275 (1997).

29. Konishi, H. et al. Activation of RAC-protein kinase by heat shock andhyperosmolarity stress through a pathway independent of phosphatidylinositol3-kinase. Proc Natl Acad Sci USA, 93, 7639-7643 (1996).

30. Dimmeler, S. et al. Activation of Nitric Oxide Synthase in Endothelial Cellsby Akt- Dependent Phosphorylation. Nature, 399, 601-605 (1999).

31. Dimmeler, S. and Zeiher, A. M. Akt Takes Center Stage in AngiogenesisSignaling. Circ Res, 86, 4-5 (2000).

32. Puck, T. T. et al. Genetics of somatic mammalian cells III. Long termcultivation of euploid cells from human and animal subjects. J. Exp. Med. 108,945–956 (1958).

33. Kao, F. T. and Puck, T. T. Genetics of somatic mammalian cells. IV.Properties of Chinese hamster cell mutants with respect to the requirements forproline. Genetics, 55, 513–524 (1967).

34. Kao, F. T. and Puck, T. T. Genetics of somatic mammalian cells, VII.Induction and isolation of nutritional mutants in Chinese hamster cells. Proc.Natl. Acad. Sci. USA 60, 1275–1281 (1968).

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35. Hansen, B. F. et al. Sustained signalling from the insulin receptor afterstimulation with insulin analogues exhibiting increased mitogenic potency.Biochem. J. 315, 271–279 (1996).

36. Flintoff, W. F. et al. Isolation and partial characterization of threemethotrexate-resistant phenotypes from Chinese hamster ovary cells.Somatic Cell Genet. 2, 245–261 (1976).

37. Ryser, H. J. and Shen, W. C. Conjugation of methotrexate to poly (L-lysine) as a potential way to overcome drug resistance. Cancer 45,1207–1211 (1980).

38. Kjeldsen, T. et al. The ligand specifities of the insulin receptor and theinsulin-like growth factor I receptor reside in different regions of a commonbinding site. Proc. Natl. Acad. Sci. USA 15, 4404–4408 (1991).

39. Zhang, J. H. et al. A Simple Statistical Parameter for Use in Evaluationand Validation of High Throughput Screening Assays. J. Biomol. Screen. 4,67–73 (1999).

40. Freshney, R. I. Cloning and Selection of Specific Cell Types in Culture ofAnimal Cells, 3rd Edition, Wiley-Liss Inc, Chapter 11, pp. 161–178 (1994).


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Chapter 10. Related products

10.1. Related productsProduct Name: Code:

GFP Assays GFP-PLCδ -PH domain assay See below*GFP-Rac1 assay See below* GFP-MAPKAP-k2 assay See below* EGFP-2xFYVE assay See below*

*Use of the GFP assays in limited as stated in the terms and conditions ofsale. The product codes vary accordingly. Please contact your localrepresentative for details.

CypHer pCORON 1000 VSV-G Expression Vector 25-8008-51pCORON 1000 SP VSV-G tag Expression Vector 25-8009-92CypHer5 Labelled Anti VSV-G Antibody PA45407 CypHer5 NHS Ester (1 mg pack) PA15401 CypHer5 NHS Ester (5 mg pack) PA15405

IN Cell Analysis System IN Cell Analyzer 3000 25-8010-11Plasma Membrane Spot Analysis Module 63-0048-96IN Cell Analyzer 1000 25-8010-26

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Chapter 11. Appendix

11.1. Appendix A: Restriction map of pCORON1000 AKT1-EGFP The following enzymes do not cut the vector: AccIII, AgeI, BseAI, BsiWI,BspEI, Bst11071 BstEII, Eco47III, EcoRV, KspI, MroI, NruI, PacI, PinAI,PmeI, SacII, SgrAI, SwaI, XbaI

Enzyme ## of cuts Positions (c) indicates the complementary strand

AatI 1 4550

AatII 5 279 332 415 601 5802

Acc65I 1 4201

AccI 1 2555

AciI 94 129 212 240 252 266 399 433 524(c) 557(c) 669 690(c) 767(c) 1178(c) 1232(c) 1236 1310(c) 1337 1397(c) 1414 1541 1631 1709 1775 1862 2041(c) 2143(c) 2210 2217 2290(c) 2804 2845 2912 2951 3089 3202 3262 3265 3307(c) 3311 3588 3649(c) 3663(c) 3666(c) 3694 3721 4099(c) 4125(c) 4138 4146(c) 4214(c) 4399 4411 4420 4432 4442 4453 4499 4654 4717 4811(c) 4875(c) 4976(c) 4979(c) 5219 5259(c) 5264 5314(c) 5330 5356 5412(c) 5471 5543 5581 5607 5617 5656 5830(c) 5877 5976(c) 6085(c) 6162(c) 6206 6327(c) 6373 6564(c) 6655(c) 7017 7026(c) 7161 7271(c) 7392(c) 7411(c) 7538(c) 7566(c)

AcsI 4 1097 3396 4050 4061

AcyI 9 276 329 412 598 2305 4745 5447 5799 6181

AflII 4 829 848 1051 4599

AflIII 3 1103 1838 2356

AluI 36 728 759 834 1048 1295 1574 1814 1940 2180 2189 2372 2606 2639 2711 2744 2960 3008 3119 3293 3435 3780 4037 4227 4515 4569 4851 5309 5670 5689 6368 6431 6531 7052 7309 7355 7445

Alw44I 3 5552 6049 7295

AlwI 24 1598(c) 1643(c) 2016 2201(c) 2558(c) 2571 3080(c) 3279 3553(c) 3562 4156 4924 4989(c) 5170 5534(c)5547 6082 6086(c) 6403 6866(c) 6867 6963(c) 6965 7051

AlwNI 2 1503 7200

AosI 4 3616 4155 4847 6498

Apal 1 1862

ApaLI 3 5552 6049 7295

ApoI 4 1097 3396 4050 4061

AseI 2 161 6546

AsnI 2 161 6546

Asp700 1 6121

Asp718 1 4201

AspEI 2 1472 6721

● 1


● 2


AspHI 9 730 2191 3195 4858 5048 5556 6053 6138 7299

AspI 1 4863

AsuII 1 5427

AvaI 5 1092 1831 2064 2232 2559

AvaII 11 1247 1351 1853 1931 2168 2229 2270 3242 5261 6357 6579

AviII 4 3616 4155 4847 6498

AvrII 1 4551

BamHI 2 2563 5539

BanI 11 619 977 1209 1729 2091 2621 3826 4201 4744 4779 6768

BanII 7 730 1482 1862 2191 2298 3796 5110

BbrPI 1 2357

BbsI 3 962 1308 2037

BbvI 31 821(c) 1132(c) 1326(c) 1561(c) 2245(c) 2333 2363 2554 2731(c) 2837 3121 3128 3154(c) 3157(c) 3422(c) 3629 3697 4168 4692(c) 4818 4860 4876(c) 4969(c) 5381 5676(c) 6287(c) 6678 6981(c) 7187(c)7190(c) 7280

BcgI 2 2710 6183(c)

Bc/l 1 2449

BfaI 11 154 753 1058 1087 3363 3714 4552 4606 6528 6863 7116

BfrI 4 829 848 1051 4599

BglI 7 137 244 366 437 3626 4504 6603

BglII 1 7621

BlnI 1 4551

BmyI 21 730 1482 1590 1862 1972 2191 2298 2626 2755 3004 3195 3796 4691 4784 4858 5048 5110 5556 6053 6138 7299

BpmI 6 1709(c) 1962 2094 3030 3270 6652

Bpu1102I 1 2373

BpuAI 3 962 1308 2037

BsaAI 4 494 2357 3867 5049

BsaBI 3 2454 3553 5538

BsaHI 9 276 329 412 598 2305 4745 5447 5799 6181

BsaI 2 916(c) 6655

BsaJI 29 514 1314 1522 1675 1831 1849 1891 2065 2233 2299 2308 2392 2559 2584 2614 2754 2917 2941 2996 4162 4263 4335 4458 4493 4502 4551 4908 5177 7449

BsaWI 5 1471 4776 6425 7256 7403

BsgI 5 1131(c) 1887(c) 2714(c) 2811 3135

BsiEI 13 665 1236 1476 1775 2147 2574 2578 3311 3597 4654 6203 6352 7275

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● 3


BsiHKAI 9 730 2191 3195 4858 5048 5556 6053 6138 7299

BsiYI 26 203 1256 1427 1613 1658 1832 1855 1907 1940 1955 2201 2281 2573 2755 2918 3268 3648 3974 4459 4726 5270 5683 7131 7410 7576 7594

BslI 26 203 1256 1427 1613 1658 1832 1855 1907 1940 1955 2201 2281 2573 2755 2918 3268 3648 3974 4459 4726 5270 5683 7131 7410 7576 7594

BsmAI 11 588 826 916(c) 941(c) 1377(c) 2400(c) 4596 5684 5726(c) 5879(c) 6655

BsmFI 12 329 480 648 1505(c) 1814(c) 1944 2215(c) 4245(c) 4317(c) 4381(c) 4896 5428

BsmI 2 3372 3465(c)

Bsp120I 1 1858

Bsp1286I 21 730 1482 1590 1862 1972 2191 2298 2626 2755 3004 3195 3796 4691 4784 4858 5048 5110 5556 6053 6138 7299

BspDI 2 3557 5526

BspHI 3 5776 5881 6889

BspMI 5 878(c) 1909 4632(c) 5013 5463

BspWI 47 137 244 366 398 437 530 554 803 1054 1292 1316 1594 1870 2186 2218 2255 2677 2737 2750 2794 2803 3596 3626 3658 3660 3702 3729 3759 4296 4368 4419 4498 4504 4736 4820 4843 4982 4988 5105 5141 5188 5455 5551 6603 6991 7563 7611

BsrBI 7 1232 1310 1397 3723(c) 5358(c) 5412 5879(c)

BsrDI 4 66(c) 4978 6487 6661(c)

BsrFI 8 2337 2569 2573 2736 3762 5064 5245 6636

BsrGI 2 97 3295

BsrI 21 449(c) 887 940 1034(c) 1486(c) 2109 2522(c) 2551(c) 3197(c) 3956 4436(c) 4689 4890 6076 6246(c) 6515 6558 6676 7082 7194(c) 7207(c)

BssHII 2 2093 5142

BstBI 1 5427

BstN1 23 244 437 1175 1524 1608 1727 1893 2058 2166 2394 2417 2631 2756 2868 2943 2997 4265 4320 4337 5132 7450 7463 7584

BstUI 25 214 1105 1543 1711 2095 2219 2914 3232 3639 3663 3683 4059 4146 4811 5112 5144 5545 5625 5728 5730 5830 6162 6655 6985 7566

BstXI 1 5466

BstYI 15 1648 2206 2563 3085 4148 4916 5162 5539 6074 6091 6859 6871 6957 6968 7621

Bsu361 1 1390

Cel/II 1 2373

Cfo/1 36 1286 1875 2095 2097 2221 2506 2875 2916 3232 3617 3641 3654 3663 3685 3711 3719 4156 4739 4747 4811 4848 5114 5144 5146 5374 5627 5730 5830 6162 6499 6592 6985 7094 7268 7368 7435

Cfr101 8 2337 2569 2573 2736 3762 5064 5245 6636

ClaI 2 3557 5526

Csp45I 1 5427

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Csp61 21 98 372 452 485 536 701 1063 1163 1565 1754 1796 1925 2054 2129 2360 3014 3296 4202 5050 55636239

DdeI 16 1390 1496 1876 2050 2373 3189 3207 4209 4511 5408 5559 5794 6220 6760 6926 7335

DpnI 37 664 749 1605 1650 1980 2010 2208 2316 2451 2565 3087 3235 3273 3556 3560 3596 4150 4918 4996 5077 5086 5164 5541 6040 6076 6093 6351 6397 6415 6756 6861 6873 6951 6959 6970 7045 7623

DpnII 37 662 747 1603 1648 1978 2008 2206 2314 2449 2563 3085 3233 3271 3554 3558 3594 4148 4916 4994 5075 5084 5162 5539 6038 6074 6091 6349 6395 6413 6754 6859 6871 6949 6957 6968 7043 7621

DraI 4 3512 6143 6835 6854

DraII 4 1931 2270 2509 5741

DraIII 3 1488 1544 3870

DrdI 6 818 3914 4588 4772 5638 7507

DsaI 5 514 2584 4162 4458 5177

DsaV 40 242 435 1173 1314 1522 1606 1725 1830 1831 1855 1891 1927 2056 2164 2392 2415 2558 2559 2613 2629 2754 2866 2941 2995 3267 3561 4263 4318 4335 4747 4907 5130 5647 5682 6183 6534 7230 7448 7461 7582

EaeI 18 9 63 1179 1233 1525 1672 2536 2548 2571 2657 3046 3308 4651 4825 5216 5243 5468 6328

EagI 4 1233 2571 3308 4651

Eam1105I 2 1472 6721

EarI 5 1501(c) 3575(c) 5089(c) 5299(c) 5922(c)

Ecl136II 2 728 2189

EclXI 4 1233 2571 3308 4651

Eco57I 10 1525 1590 1770 2739 2783(c) 2982 4891 5323 6055 7067(c)

EcoNI 3 1611 1656 2199

EcoO109I 4 1931 2270 2509 5741

EcoRI 1 1097

EcoRII 23 242 435 1173 1522 1606 1725 1891 2056 2164 2392 2415 2629 2754 2866 2941 2995 4263 4318 4335 5130 7448 7461 7582

Esp3I 2 5684 5726(c)

EspI 1 2373

Fnu4HI 59 835 1146 1179 1233 1236 1311 1340 1575 1631 2042 2259 2322 2352 2543 2745 2804 2826 3110 3117 3168 3171 3265 3308 3311 3436 3618 3650 3664 3686 4157 4499 4654 4706 4717 4807 4812 4849 4890 4977 4980 4983 5219 5315 5356 5370 5471 5581 5690 5977 6206 6301 6328 6667 6995 7201 7204 7269 7412 7567

FnuDII 25 214 1105 1543 1711 2095 2219 2914 3232 3639 3663 3683 4059 4146 4811 5112 5144 5545 5625 5728 5730 5830 6162 6655 6985 7566

FokI 14 984(c) 1254 1320(c) 1404(c) 2178(c) 2613(c) 2979(c) 4402(c) 5069 5094 5639(c) 6282 6569 6750

FspI 4 3616 4155 4847 6498

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HaeII 5 1876 3712 3720 4748 7369

HaeIII 48 11 65 238 431 1181 1235 1313 1319 1438 1527 1623 1630 1674 1689 1860 2061 2238 2442 2510 2538 2550 2573 2659 2759 3048 3160 3310 3586 3875 4017 4167 4492 4498 4507 4550 4653 4827 5218 5245 5470 5743 6330 6597 6677 7135 7569 7587 7598

HgaI 10 688 1094(c) 1700(c) 2313 3645 5455 5631 6189 6919(c) 7497(c)

HgiAI 9 730 2191 3195 4858 5048 5556 6053 6138 7299

HhaI 36 1286 1875 2095 2097 2221 2506 2875 2916 3232 3617 3641 3654 3663 3685 3711 3719 4156 4739 4747 4811 4848 5114 5144 5146 5374 5627 5730 5830 6162 6499 6592 6985 7094 7268 7368 7435

HinP1I 36 1284 1873 2093 2095 2219 2504 2873 2914 3230 3615 3639 3652 3661 3683 3709 3717 4154 4737 4745 4809 4846 5112 5142 5144 5372 5625 5728 5828 6160 6497 6590 6983 7092 7266 7366 7433

HincII 3 678 2556 3451

HindII 3 678 2556 3451

HindIII 3 757 2178 4567

HinfI 14 564 842 958 1074 1385 3915 3937 4573 5230 5364 5416 5523 6722 7239

HpaI 1 3451

HpaII 34 1316 1472 1832 1856 1928 2338 2560 2570 2574 2614 2677 2737 3268 3562 3763 4650 4727 4749 4777 4908 4998 5065 5246 5649 5683 6184 6426 6536 6603 6637 7041 7231 7257 7404

HphI 21 530 1100(c) 1474(c) 1613 1622 1691(c) 2444(c) 2600 2603(c) 2933 2957 3086 3867 4923(c) 5701(c) 5710(c) 5994(c) 6029 6235(c) 6651 6878

ItaI 59 835 1146 1179 1233 1236 1311 1340 1575 1631 2042 2259 2322 2352 2543 2745 2804 2826 3110 3117 3168 3171 3265 3308 3311 3436 3618 3650 3664 3686 4157 4499 4654 4706 4717 4807 4812 4849 4890 4977 4980 4983 5219 5315 5356 5370 5471 5581 5690 5977 6206 6301 6328 6667 6995 7201 7204 7269 7412 7567

KasI 1 4744

KpnI 1 4205

Ksp632I 5 1501(c) 3575(c) 5089(c) 5299(c) 5922(c)

MaeI 11 154 753 1058 1087 3363 3714 4552 4606 6528 6863 7116

MaeII 26 75 276 288 329 412 493 598 1120 1253 1838 1918 2356 2400 2650 2863 3034 3756 3866 3909 3921 4861 5048 5799 6119 6492 6908

MaeIII 25 215 302 651 839 902 1106 1488 1544 2396 2768 3257 3421 3677 3689 4865 5171 5672 6060 6248 6401 6459 6790 7073 7189 7252

MamI 3 2454 3553 5538

MboI 37 662 747 1603 1648 1978 2008 2206 2314 2449 2563 3085 3233 3271 3554 3558 3594 4148 4916 4994 5075 5084 5162 5539 6038 6074 6091 6349 6395 6413 6754 6859 6871 6949 6957 6968 7043 7621

MboII 25 967 1313 1518 1658 1811(c) 1925 2042 2378 2828(c) 2873(c) 2876(c) 3071 3592 3728(c) 4568(c) 5106 5316 5396(c) 5939 6048 6126 6881 6952(c) 7104(c) 7616(c)

McrI 13 665 1236 1476 1775 2147 2574 2578 3311 3597 4654 6203 6352 7275

MfeI 1 3460

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MluI 1 1103

MluNI 5 11 65 1527 1674 4827

MnlI 67 703(c) 870(c) 1101 1130(c) 1149(c) 1203 1206 1250(c) 1275 1385(c) 1394(c) 1445(c) 1448(c) 1449 1451(c) 1508(c) 1662 1676(c) 1749 1788 1844(c) 1944 1959 1997(c) 2060(c) 2069(c) 2195(c) 2205 2228(c) 2275(c) 2294(c) 2423(c) 2594(c) 2675(c) 2681(c) 2775 2912(c) 2924(c) 2975(c) 3095(c) 3496(c) 3536 3576(c) 3840 4180(c) 4188 4204(c) 4482(c) 4488(c) 4512 4518 4525(c) 4528(c) 4540(c) 4660(c) 4796(c) 5153(c) 5346 5695(c) 5754 6348(c) 6554(c) 6701 6782 7182 7432(c) 7506

MscI 5 11 65 1527 1674 4827

MseI 28 161 784 830 849 917 1052 1067 1975 3450 3511 3657 3928 4026 4043 4054 4066 4077 4600 5589 5770 6142 6507 6546 6781 6834 6848 6853 6905

MslI 13 519 2196 2586 2616 2766 2943 3072 5182 5464 5503 5950 6309 6468

MspA1I 8 1295 1339 4227 4851 5619 6085 7026 7271

MspI 34 1316 1472 1832 1856 1928 2338 2560 2570 2574 2614 2677 2737 3268 3562 3763 4650 4727 4749 4777 4908 4998 5065 5246 5649 5683 6184 6426 6536 6603 6637 7041 7231 7257 7404

MunI 1 3460

MvaI 23 244 437 1175 1524 1608 1727 1893 2058 2166 2394 2417 2631 2756 2868 2943 2997 4265 4320 4337 5132 7450 7463 7584

MvnI 25 214 1105 1543 1711 2095 2219 2914 3232 3639 3663 3683 4059 4146 4811 5112 5144 5545 5625 5728 5730 5830 6162 6655 6985 7566

MwoI 47 137 244 366 398 437 530 554 803 1054 1292 1316 1594 1870 2186 2218 2255 2677 2737 2750 2794 2803 3596 3626 3658 3660 3702 3729 3759 4296 4368 4419 4498 4504 4736 4820 4843 4982 4988 5105 5141 5188 5455 5551 6603 6991 7563 7611

NaeI 2 3764 5247

NarI 1 4757

NciI 17 1316 1832 1833 1857 1929 2560 2561 2615 3269 3563 4749 4909 5649 5684 6185 6536 7232

NcoI 5 514 2584 4162 4458 5177

NdeI 1 388

NdeII 37 662 747 1603 1648 1978 2008 2206 2314 2449 2563 3085 3233 3271 3554 3558 3594 4148 4916 4994 5075 5084 5162 5539 6038 6074 6091 6349 6395 6413 6754 6859 6871 6949 6957 6968 7043 7621

NgoMI 2 3762 5245

NheI 1 1086

NlaIII 41 118 136 458 518 1115 1380 1553 1646 1793 1955 2030 2129 2175 2201 2321 2486 2588 2822 2852 3047 3242 3287 3328 4166 4299 4371 4462 4619 4964 5150 5181 5207 5696 5780 5885 6278 6314 6392 6402 6893 7613

NlaIV 33 621 979 1211 1261 1314 1731 1860 1932 2021 2062 2231 2272 2297 2511 2565 2623 3161 3795 3807 3828 4203 4269 4341 4746 4781 5541 5834 6424 6635 6676 6770 7542 7581

NotI 2 1233 3308

NsiI 2 4301 4373

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NspI 4 4299 4371 5150 5696

NspV 1 5427

PaeR7I 1 1092

Pf/MI 1 1427

PleI 10 558(c) 836(c) 952(c) 1068(c) 1379(c) 3923 3931(c) 5410(c) 6730 7233(c)

PmaCI 1 2357

PmlI 1 2357

Ppu10I 2 4297 4369

PpuMI 2 1931 2270

Psp1406I 2 6119 6492

PstI 4 839 1348 1720 4798

PvuI 3 665 3597 6352

PvuII 3 1295 4227 4851

RcaI 3 5776 5881 6889

RsaI 21 99 373 453 486 537 702 1064 1164 1566 1755 1797 1926 2055 2130 2361 3015 3297 4203 5051 55646240

RsrII 1 5261

SacI 2 730 2191

SalI 1 2554

SapI 2 5089(c) 5299(c)

Sau3AI 37 662 747 1603 1648 1978 2008 2206 2314 2449 2563 3085 3233 3271 3554 3558 3594 4148 4916 4994 5075 5084 5162 5539 6038 6074 6091 6349 6395 6413 6754 6859 6871 6949 6957 6968 7043 7621

Sau96I 28 237 430 1247 1312 1318 1351 1688 1853 1858 1859 1931 2060 2168 2229 2270 2441 2509 2758 3159 3242 3585 3873 5261 5741 6357 6579 6596 6675

ScaI 3 1064 1755 6240

ScrFI 40 244 437 1175 1316 1524 1608 1727 1832 1833 1857 1893 1929 2058 2166 2394 2417 2560 2561 2615 2631 2756 2868 2943 2997 3269 3563 4265 4320 4337 4749 4909 5132 5649 5684 6185 6536 7232 7450 7463 7584

SexAI 2 2415 4318

SfaNI 23 511(c) 2333 2684(c) 2962 2977 3076 3394(c) 4086(c) 4126 4308 4380 4703(c) 4958(c) 5044 5108 5174(c) 5383 5567(c) 5661 6020(c) 6269 6460(c) 7512(c)

SfcI 9 835 1080 1344 1716 3644 4794 6475 7153 7344

SfiI 1 4504

SfuI 1 5427

SmaI 2 1833 2561

SnaBI 1 494

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SnoI 3 5552 6049 7295

SpeI 1 153

SphI 3 4299 4371 5150

SspBI 2 97 3295

SspI 4 6 53 4075 5916

StuI 1 4550

StyI 8 514 1675 2308 2584 4162 4458 4551 5177

TaqI 24 824 945 1093 1363 2555 2635 2929 2956 2971 3100 3316 3557 3832 4594 4858 5014 5038 5074 5236 5427 5526 6067 7511 7616

TfiI 4 4573 5230 5364 5523

ThaI 25 214 1105 1543 1711 2095 2219 2914 3232 3639 3663 3683 4059 4146 4811 5112 5144 5545 5625 5728 5730 5830 6162 6655 6985 7566

Tru9I 28 161 784 830 849 917 1052 1067 1975 3450 3511 3657 3928 4026 4043 4054 4066 4077 4600 5589 5770 6142 6507 6546 6781 6834 6848 6853 6905

Tsp509I 15 172 786 1040 1097 3396 3460 4050 4061 4087 4305 4377 4469 6288 6543 6849

Tth111I 1 4863

Van91I 1 1427

XcmI 3 1118 1424 1682

XhoI 1 1092

XhoII 15 1648 2206 2563 3085 4148 4916 5162 5539 6074 6091 6859 6871 6957 6968 7621

XmaI 2 1831 2559

XmaIII 4 1233 2571 3308 4651

XmnI 1 6121

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