10amOverviews..

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10am Overviews.. Gateway technology Expressi
on systems 11 am Entry methods Noon Lunch
putting it all together Overview of
Freezer set-up Inventory management
Invitrogen Support 1 Vector conversion
simplifed 145 - 3 pm Open session -
consultation 3 4 pm Gateway BLOCK-iT for RNAi
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Overviews Gateway technology Expression
systems

• U Penn School of Dental Medicine
• March 15, 2005
• Dolores Ciufo
• Technology Specialist

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Gateway technology overview

• Why develop a new cloning technology?
• What Gateway can do for you
• Gateway basics

Expression systems

• Tags and proteases
• BioEase
• Lumio
• Expression systems
• Cell-free
• Prokaryotic
• Insect
• Mammalian

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Multiple Genome Sequencing Projects Global Gene
Expression Analysis (microarrays)
Proteomics Functional Genomics
Optimization of protein purification
Structure analysis crystalization
Functional assays
Expression in multiple cellular contexts
Subcellular localization
A new cloning technology is needed
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What if..?

• Maximize compatibility and flexibility
  universally compatible vector system
• Minimize planning eliminate restriction site
  selection problems
• Maintain reading frame and orientation
• Rapid
• No restriction enzymes
• No gel purification
• No ligations
• High-throughput

A
B
A better way to clone - Gateway
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Gateway cloning technology is.

• Site-specific recombination - one hour subcloning
  reaction eliminates RE, ligase and colony
  screening
• Transfers one or more genes into one or more
  expression vectors in one experiment
• Maintains reading frame and orientation
• High efficiency - gt90, typically gt99, desired
  clones
• Any vector can be made Gateway - compatible
• Simple, fast, robust reactions - automatable

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Gateway Platform
Gateway Technology is central for protein
analysis
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Gateway Platform what it can do for you
Reduce cloning time from months to days Rapidly
move from one application to the next Accelerate
your research via key collaborations Easy access
to new technology Lumio BioEase GeneBLAzer
RNAi Lentivirus Adenovirus Consider new
approaches to research large scale, HTP
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EMBO Reports
Gateway enabled project
Taken from EMBO reports,1, p.287, 2000
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December 8, 2003  PFGRC announces Gateway Clone
Sets The National Institute of Allergy and
Infectious Diseases (NIAID) sponsored Pathogen
Functional Genomics Resource Center (PFGRC) at
The Institute for Genomic Research (TIGR) is
planning on making available, at no cost to the
research community, recombinant entry clones of
full length open reading frames (ORFs)
constructed in Invitrogen's Gateway Cloning
Technology. S. pneumoniae, B. anthracis, Y.
pestis, and M. tuberculosis
December 8, 2003  SARS-CoV Gateway Clone Set
available The National Institute of Allergy and
Infectious Diseases (NIAID) sponsored Pathogen
Functional Genomics Resource Center (PFGRC) at
The Institute for Genomic Research (TIGR) has
constructed and is making available a clone set
called the SARS-CoV Gateway Clone Set. This
clone set consists of twenty seven (27) clones
containing ORFs based on the Genbank accession
number NC_004718.3. Each clone in the set is
clearly identified by its accession. The clones
are constructed in the vector pDONR 221,
(Invitrogen, Inc). These "entry clones" are
constructed such that in-frame expression
constructs can be readily produced through the
use of Invitrogen's Gateway Cloning Technology.
Recognized platform technology
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Publications Referencing Gateway
Over 400 citations in 2004
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Gateway Cloning Technology
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Phage ? recombination in E.coli
Conservative Recombination
Phage l
att P
att B
E. coli
Excision
Integration
att L
att R
Integrated prophage
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Direction control in recombination

• To obtain directionality in cloning/subcloning
• the gene must be flanked with different
  recombination sites
• the sites must have specificity for only their
  partner

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Gateway cloning technology

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Terminology
Entry clones att L DESTination vectors att
R DONR vectors att P Expression
clones att B
You Enter your hotel through the Lobby and
Rest at your DESTination.
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LR Clonase II Reaction Protocol

• Entry Clone (50-150 ng) 1-7 µl
• Destination Vector (150 ng/µl) 1 µl
• TE to adjust volume to 8 µl
• LR Clonase II Enzyme 2 µl
• 1. Mix and incubate for 60 min at Room Temp
  (25oC).
• 2. Add Proteinase K solution and incubate for 10
  min at 37oC.
• 3. Transform competent E. coli with 1 µl per 50
  µl cells.
• 4. Express for 60 minutes and plate on LB-Amp.

Simple, fast reactions
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Subcloning example
CAT gene
Gene
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High efficiency transfer
Expression Vector Colonies Background
Analysis Native (E. coli) 15,000 0
4/4 6xHis-fusion 10,650 0
4/4 GST-fusion 9,200 0
4/4 Thioredoxin-fusion 11,000 0
4/4 Sequencing Strand 13,950 0
4/4 Sequencing - Strand 8,950 0
4/4 Native protein (baculo) 7,800 15
4/4 6xHis (baculo) 6,350 30
4/4 CMV-promoter 7,950 0
4/4 Tet-regulated promoter 6,350 0
4/4
(Less than 1)
Why would you want to do multiple transfers?
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Optimization of protein expression

• Selection of best
• host/vector system
• Selection of the best tag
• His, GST, Myc, HA, Flag
• Selection of position
• N-terminal
• C-terminal

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Expression of full-length human ORFs
BL21-SI
Sf9 Cells
lt
Human-MAP4
50 kDa
gt
Human-EIF4e
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Expression Screening in E. coli
Rapid screening for improved solubility of small
human proteins produced as fusions in E.coli
Tags can make a difference
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Expression screening in E.coli

• Detectable over expression from 96 (26/32)
• Soluble expression from 85 with at least one tag
  (23/32)

Include several fusion tags in expression
solubility screening
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Recombinant protein expression
Eukaryotic expression challenges

• Some cell lines and tissues are hard to transfect
  for recombinant gene expression consider viral
  transduction
• Traditional viral systems can be difficult to
  work with Gateway technology addresses this
  directly
• Mammalian regulated expression systems are
  typically leaky T-REx, Tetracycline Regulated
  Expression
• No one system can successfully express every
  eukaryotic protein be prepared to use several
  vectors/tags

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Gateway technology overview

• Why develop a new cloning technology?
• What Gateway can do for you
• Gateway basics

Expression systems

• Tags and proteases
• BioEase
• Lumio
• Expression systems
• Cell-free
• Prokaryotic
• Insect
• Mammalian

The quick tour
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Different Expression Systems
Cellular Analysis
Protein Production
Easiest to Use
Cost of media and equipment
PTM / Probability of protein function
Time Requirement
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Fusion tags protease cleavage sites
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BioEase Tag

• 72 aa tag that is biotinylated in vivo in E.
  coli, insect cells, and mammalian cells
• A single biotin will be incorporated into the
  protein at the N-terminal end
• There is a very high affinity, specific, and
  irreversible bond between biotin and streptavidin
• EK Cleavage used to cleave off the Biotag from
  the protein

• Use for
• Protein purification
• Protein-protein interactions
• Protein complex purification
• Immunofluorescent intracellular detection

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Purification of Biotinylated ?-Gal
Efficient Tag Removal w/ EKMax
Easy Detection and Purification
ß-Gal
Flow thru increasing O/N incubations w/ EKMax
Lysate
Wash Fractions
Flow thru after incubation w/ resin
Flow thru O/N no EK Max
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Lumio technology

• Sensitive localization of proteins in living
  cells
• Real-time monitoring of protein expression
• Fast, easy in-gel detection of proteins
• Compatible with downstream applications

Lumio tag 6 aa tag with a tetracysteine motif
(C-C-X-X-C-C) rarely occurring motif binds
Lumio reagent Lumio reagent non-fluorescent
bi-arsentical reagent binds Lumio tag sequence
? fluorescence
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Lumio reagent expression vectors
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Lumio Real Time Detection
Human ORFs
50 ul Expressway reaction in 96 well plate, 37C
incubation in SPECTRAmax GeminiXS plate reader.
Ex 500 nm monitored Em 535 nm. Reading every 10
min over 1 hr period
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Lumio and Western Detection
Expressway
Visualization of Lumio-tagged Kinases. Kinases
from Invitrogens Human Orf Collection were
expressed from the pEXP3 vector. One microliter
of the Expressway HTP reaction was analyzed on
an E-PAGE? 96 6 gel using Lumio Green sample
buffer and detection reagent. Bands were
visualized on a Typhoon Laser scanner. No DNA in
wells B4, C8, D7, E3, E4. Cat control in Lane
H12.
BenchMark Fluorescent Protein Standard (11
155 kDa, ready-to-use, pre-labeled with
fluorescent dye for molecular weight sizing)
Western Blot of His-tagged Kinases. After
visualization on the laser scanner, the proteins
were transferred to PVDF membrane for Western
Blot. A 14000X of Anti-HisG-HRP was used in the
Invitrogens WesternBreeze Chemiluminescent Kit.
NOTE Always use the Lumio Gel Sample buffer to
prep gel samples
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Lumio tag visualization
Coomassie stain
Laser scanner
UV light box (305 or 365nm)
Ln 1-3 CAT Ln 4-6 GFP Ln 7-9 GUS Ln 10
no DNA
E.coli expressed proteins
Tips Standard SDS-PAGE sample buffer not
compatible Green fluorescent signal
stable for 10-15 min Document before
further processing
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Expression Systems tags
Tags Protease sites
Expression systems ? Expressway Cell Free
expression Prokaryotic ? BaculoDirect
Insect Mammalian Tag-on-Demand Lumio N
C terminal GFP Flp-In TRex ViraPower
Lentivirus ViraPower Adenovirus
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Expressway Plus
Cell-free expression

• E. coli extract that allows simultaneous
  transcription translation
• Produce full-length, functional protein in just
  2 hours
• Eliminate the hassle and cost of cell
  maintenance
• Scale the reaction to achieve microgram to
  milligram levels of protein
• Real-time detection of protein production with
  Lumio tag technology

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How Expressway? Works

• Generate DNA template (Linear or plasmid)
• Purify DNA template (PureLink miniprep or PCR
  cleanup kit)
• Add Expressway component. Incubate for 2 hours
  (4 hr incub ? 20-50 more protein)
• Analyze sample
• Coomassie stain
• Western blot analysis
• Assay activity

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Expressway Vectors
pEXP1- DEST N-term 6xHis / Xpress- EK pEXP2-
DEST C-term V5-6xHis pEXP3- DEST N-term Lumio /
Tev
Expressway lysate formats Expressway
Plus Expressway HTP Expressway TAG-on-Demand
(includes suppressor tRNA mix)
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Anatomy of the BaculoDirect System
Linear BaculoDirect DNA
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BaculoDirect
Baculovirus expression faster and easier
than ever!
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BaculoDirect Vector options
BaculoDirect C-term BaculoDirect
N-term BaculoDirect Secreted
C-term V5-His N-term His-V5-TEV N-term Melittin
secretion signal His-V5,TEV
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Expression Systems tags
Tags Protease sites
Expression systems Expressway Cell Free
expression Prokaryotic BaculoDirect
Insect Mammalian ? Tag-on-Demand Lumio
N C terminal GFP Flp-In
TRex ViraPower Lentivirus ViraPower
Adenovirus
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tRNA Suppression Tag-on-Demand
Mammalian cells Adenoviral-based stop
suppression technology Cell Free Bacteriophage
T4 amber suppressor tRNA Efficient delivery to
a large variety of mammalian cells Allows native
or C-term tagged expression from a single
expression vector Detect expression or
localization of a recombinant protein when no
antibody is available
Available vectors pcDNA6.2/V5-DEST
pcDNA6.2/GFP-DEST
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tRNA Suppression Tag-on-Demand
Compatible with entry clones containing TAG stop
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Tag-On-Demand ORF localization
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Expression Systems tags
Tags Protease sites
Expression systems Expressway Cell Free
expression Prokaryotic BaculoDirect
Insect Mammalian Tag-on-Demand Lumio N
C terminal GFP ? Flp-In
? TRex ViraPower Lentivirus ViraPower
Adenovirus
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Flp-In Targeted Integration
System Benefits

• Site-specific integration

• Rapid generation of a homogeneous population
• a single transfection
• no clonal isolation
• No chromosomal position effects
• Permits direct comparisons of different
  constructs (e.g. promoters, gene families, etc.)
• Ideal for high-throughput expression studies

Generate Isogenic Expression Lines
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To Make a Flp-In host cell line
Available pre-made Flp-In cell
lines Flp-In-293 Flp-In-Jurkat Flp-In-CV-1 Fl
p-In-CHO Flp-In-BHK Flp-In-3T3 Flp-In/T-REx-29
3
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Generating Flp-In expression cells
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Flp-In vs. traditional stable transfection
Flp-In CHO cells
Standard Transfection (pcDNA5/CAT)
Flp-In Transfection (pcDNA5/FRT/CAT)
No need to isolate single clones Directly compare
results
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T-REx Tetracycline-Inducible System
TetR expressing Cell line

• CMV promoter expression regulated by tetracycline
• Expression of integrated gene can be either
• - constitutive (CMV) in regular cell lines
• - inducible (tetracycline) in T-REx Cell
  Lines
• No leaky minimal promoters, no viral
  transactivators
• T-Rex cell lines available 293, HeLa, CHO and
  Jurkat

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Induced lacZ expression
T-REx CHO lacZ stable cells
T-REx 293 cells stably expressing pcDNA4/TO/lacZ
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Flp-In and T-REx vectors
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Expression Systems tags
Tags Protease sites
Expression systems Expressway Cell Free
expression Prokaryotic BaculoDirect
Insect Mammalian Tag-on-Demand Lumio N
C terminal GFP Flp-In TRex
? ViraPower Lentivirus ? ViraPower
Adenovirus
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Selecting a viral expression system
Transient Expression
Stable Expression
Dividing Cells
Non- Dividing Cells
Dividing Cells
Neuronal Cells
Drug or Growth Arrested Cells
Contact Inhibited Cells
 
 
 
 
Adenovirus (DNA virus)
YES
YES
 
 
 
 
Retrovirus (RNA virus)
YES
YES
Lentivirus (RNA virus)
YES
YES
YES
YES
YES
YES
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Lentiviral System Introduction

• RNA retrovirus
• Based on HIV-1, but only uses gag/pol and rev
• Third generation SIN vectors (Naldini and
  Verma)
• Active nuclear import (in contrast to traditional
  murine retroviral systems)
• Efficient transduction of hard-to-transfect cells

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Virus Production and Transduction
Deliver to non-dividing, hard to transfect cells
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ViraPower Lentiviral Production
Optimized ViraPower Packaging Mix
pLenti Vector with GOI
Gag/pol Required for packaging
Add to mammalian cells to transduce
Rev Added safety feature
48 hours
VSV-G envelope Broad Tropism

Store up to 1 year
Virus secreted into media
293FT Cell Line

Lipofectamine 2000 (provided in the ViraPower
Support Kit)
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1o Human Fibroblasts
Expression in non-dividing cells
pLenti6/GFP

• 100 confluent 2 wks
• MOI 1
• 48 hr post transduction

• 12 day cultures
• MOI 2
• 72 hr post transduction

Tradnl Retroviral Vector
pLenti6/V5-lacZ Lentiviral Vector
Lenti works when standard Retroviruses cant
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Selecting a viral expression system
Transient Expression
Stable Expression
Dividing Cells
Non- Dividing Cells
Dividing Cells
Neuronal Cells
Drug or Growth Arrested Cells
Contact Inhibited Cells
 
 
 
 
Adenovirus (DNA virus)
YES
YES
 
 
 
 
Retrovirus (RNA virus)
YES
YES
Lentivirus (RNA virus)
YES
YES
YES
YES
YES
YES
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Adenovirus General Information

• Adenovirus has a 35 kb linear dsDNA genome
• ViraPower Adenoviral gene vectors are
  E1-deleted and E3-deleted (first generation)
• E1 is supplied in trans in all 293 cells
• E3 is completely dispensable for all in vitro
  applications
• E1-, E3-deleted viruses are replication
  incompetent
• (except in E1-expressing cells, e.g. 293 cells)
• Packaging limits 7.5 kb foreign DNA
• Viral genome actively transported to nucleus, but
  DOES NOT integrate in host genome

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ViraPower? Adenovirus Vectors

• Choose your promoter
• Entry clone must contain promoter and GOI

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Flowchart of adenovirus creation
Streamlined..... Fast, efficient cloning
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Recombination Efficiency and Accuracy
Gateway
Traditional
A. EFFICIENCY
B. CLONES (EcoRI digested)
100 correct (10 out of 10)
10 correct (1 out of 10)
C. ACCURACY
Screen. Dont Scream
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Expression Systems tags
Tags Protease sites
Expression systems Expressway Cell Free
expression Prokaryotic BaculoDirect
Insect Mammalian Tag-on-Demand Lumio N
C terminal GFP Flp-In TRex ViraPower
Lentivirus ViraPower Adenovirus
The whirl-wind quick-tour..
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Summary Gateway Technology

• Based on site-specific recombination, Gateway
  Technology is
• Highly efficient
• typically gt99 desired clones
• universally compatible recombinational subcloning
  vectors
• verify entry clone, but resequencing of each
  subclone is not necessary
• Simple, fast, and robust
• One-hour reactions on the benchtop
• Amenable to high-throughput
• Transfer one or more genes into multiple vectors
  in parallel experiments
• Flexible
• Any vector can be Gateway adapted
• You can easily move from one application to the
  next
• Powerful
• Enables easy manipulation of advanced gene
  expression systems

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Questions?
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New vectors technologies
BLOCK-iT RNAi Vectors
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RNAi Vectors for delivery of shRNA
dsDNA oligo
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Mechanism of tetracycline pol III regulation
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Selection of BLOCK-iTTM RNAi DEST Vectors
Traditional Selection Vectors
Viral Vectors
Lots of options!
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Long-term inducible blocking of shRNA expression
Long-term,stable regulation of shRNA expression
activity
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Dose response to tetracycline
Control shRNA expression by dosage
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A bit more about ccdB ..

• The ccdB Negative Selection System
• In E. coli expression of topoisomerase II is
  lethal in the presence of a protein ccdB (control
  of cell death)
• Lethality is prevented by the ccdA protein
• Thus, expression of ccdB is lethal unless ccdA is
  present or ccdB is removed or inactivated.
• ccdA is on the F episome

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So.. how do you propagate vectors with ccdB
genes?

• What does this mean with respect to growth and
  maintenance of plasmids that contain ccdB? e.g.
  DEST and DONR vectors
• You need to maintain these plasmids in an E. coli
  strain that can tolerate this lethal gene.
• One-Shot ccdB Survival T1-Phage-Resistant cells
  have a gyrase mutation that make them tolerant to
  ccdB.
• Take away message Dont transform Gateway
  reactions into F plasmid containing strains of
  E. coli.

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Naming clones.
You Enter your hotel through the Lobby and Rest
at your DESTination.
Entry clones att L pENTR-gene name
Destination vectors att R pDEST- vector
descriptors Donor vectors att P pDONR- or
descriptor Expression clones att
B pEXP-DESTinfo-gene name
LR reaction examples
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BP Clonase II Reaction Protocol

• attB-PCR product (gt10 ng/ µl final amt 15-150
  ng) 1-7 µl (50 fmoles)
• Donor Vector (150 ng/µl) 1 µl (50
  fmoles)
• TE to adjust volume to 8 µl
• BP Clonase II Enzyme 2 µl
• 1. Mix and incubate for 60 min at Room Temp
  (25oC).
• 2. Add Proteinase K solution and incubate for 10
  min at 37oC.
• 3. Transform competent E. coli with 1 µl per 50
  µl cells.
• 4. Express for 60 minutes and plate on LB-Kan.

Quantitation of your entry clone is necessary
for optimal performance e.g. Use OD 260 data
and equation in Gateway manual to calculate
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How Expressway? Works

• Generate DNA template (Linear or plasmid)
• Purify DNA template (PureLink miniprep or PCR
  cleanup kit)
• Add Expressway component. Incubate for 2 hours
  (4 hr incub ? 20-50 more protein)
• Analyze sample
• Coomassie stain
• Western blot analysis
• Assay activity

79
Expressway Plus
Higher yield, more full-length, functional protein
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Proteases
AcTev Protease
EKMax Enterokinase
Tev active at 4oC to 30oC pH 6.0 to 8.5
81
Lumio Technology
Detection and localization of protein in cells
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pLenti6/V5 Expression Vectors
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GatewaySummary

• Flexible transfers DNA segments into multiple
  vectors in parallel
• gt 90 Efficiency
• Reading frame and orientation maintained
• Any expression vector can be made
  GATEWAY-compatible
• Reaction is simple, rapid, and potentially
  automatable
• Platform technology readily integrates new
  technologies

84
ViraPower Lentiviral Production
Optimized ViraPower Packaging Mix
Add to mammalian cells to transduce
Gag/pol Required for packaging
Rev Added safety feature
48 hours
VSV-G envelope Broad Tropism
Store up to 1 year

pLenti Vector with GOI
Virus secreted into media
293FT Cell Line

Lipofectamine 2000 (provided in the ViraPower
Support Kit)