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Lecture 12 - 2/15/2001 Transcription factors I
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Topics we will cover today
– transgenic technology (contd from last time)
• gene trapping
• conditional gene targeting
– regulated expression of introduced genes
• ecdysone
• tetracycline
• reverse tetracycline
• hybrid tetracycline
– implications of this technology
• genetics and reverse genetics
• clinical genetics
• gene therapy
• protein engineering
• metabolite engineering
• transgenic food
• plants as producers of specialty chemicals
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BioSci 145A lecture 12
page 1
©copyright
Bruce Blumberg 2000. All rights reserved
Gene trapping
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Observation is that various types of viruses and
transposable elements can be utilized to deliver DNA to
random locations
– this can disrupt gene function OR
– bring the inserted gene under the control of adjacent
regulatory sequences OR
– both
several flavors
– enhancer trap is designed to bring inserted reporter
gene under the control of local regulatory sequences
• typically put a reporter gene adjacent to a weak
promoter (enhancer-less), e.g. a retrovirus with
enhancers removed from the LTRs
• may or may not disrupt expression
BioSci 145A lecture 12
page 2
©copyright
Bruce Blumberg 2000. All rights reserved
Gene trapping (contd)
– enhancer trap (contd)
• expression only results when integration occurs
into an active transcription unit
• reporter expression then duplicates the temporal
and spatial pattern of the endogenous gene
• reporters used
– -gal was the most widely used reporter
– GFP is now popular
– -lactamase is seeing increasing use
• advantages
– relatively simple to perform
– active promoters seem to be frequently
targeted, perhaps due to open chromatin
• disadvantages
– insertional mutagenesis is not the goal and
does not occur with high frequency
– overall frequency is not that high
– relies on transposon or retroviruses to get
insertion
» may not be available for all systems,
requires transgenesis or good viral
vectors
BioSci 145A lecture 12
page 3
©copyright
Bruce Blumberg 2000. All rights reserved
Gene trapping (contd)
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Flavors of gene trapping (contd)
– expressed gene trap (many variations possible)
• designed to fuse inserted reporter with coding
sequences of endogenous gene
• goal is to cause loss of expression of endogenous
gene and replace it with the transgene
• typically done in ES cells to generate a library of
insertional mutagens
– also widely used in Drosophila and zebrafish
• reporter expression duplicates the temporal and
spatial pattern of the endogenous gene
• reporters used
– -gal was the most widely used reporter
– GFP is now popular
– -lactamase is seeing increasing use
BioSci 145A lecture 12
page 4
©copyright
Bruce Blumberg 2000. All rights reserved
Gene trapping (contd)
– Expressed gene trapping (contd)
• advantages
– insertional mutagen
» gives information about expression
patterns
» can be homozygosed to generate
phenotypes
– higher efficiency than original trapping
methods
– selectable markers allow identification of
mutants
» many fewer to screen
» dual selection strategies possible
• disadvantages
– overall frequency is still not that high
– frequency of integration into transcription
unit is not high either
– relies on transposon or retroviruses to get
insertion
» may not be available in your favorite
system.
BioSci 145A lecture 12
page 5
©copyright
Bruce Blumberg 2000. All rights reserved
Conditional gene targeting
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Many gene knockouts are embryonic lethal
– some of these are appropriate and expected
• gene activity is required early
– others result from failure to form and/or maintain the
placenta
• ~30% of all knockouts
How can this be overcome?
– Generate conditional knockouts either in particular
tissues or after critical developmental windows pass
– Sauer (1998) Methods 14, 381-392.
Approach
– recombinases exist that can perform site-specific
excision of sequences between recognition sites
– FLP system from yeast
• not widely used, doesn’t work well
– Cre/lox system from bacteriophage P1
• P1 is a temperate phage that hops into and out of
the bacterial genome
• recombination requires
– 34 bp recognition sites called locus of
crossover x in P1 (loxP)
– Cre recombinase
• if loxP sites are directly repeated then deletions
• if inverted repeats then inversions result
BioSci 145A lecture 12
page 6
©copyright
Bruce Blumberg 2000. All rights reserved
Conditional gene targeting - contd
BioSci 145A lecture 12
page 7
©copyright
Bruce Blumberg 2000. All rights reserved
Conditional gene targeting (contd)
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Strategy
– targeting construct (minimum needed for grant)
– homologous recombination,
– transfect CRE, select for loss of tk
– Southern to select correct event
– inject into blastocysts and select chimeras
– establish lines
– cross with Cre expressing line and analyze function
BioSci 145A lecture 12
page 8
©copyright
Bruce Blumberg 2000. All rights reserved
Conditional gene targeting (contd)
BioSci 145A lecture 12
page 9
©copyright
Bruce Blumberg 2000. All rights reserved
Conditional gene targeting (contd)
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advantages
– can target recombination to specific tissues and times
– can study genes that are embryonic lethal when
disrupted
– can use for marker eviction
– can study the role of a single gene in many different
tissues with a single mouse line
– can use for engineering translocations and inversions
on chromosomes
disadvantages
– not trivial to set up, more difficult than std ko but
more information possible
– requirement for Cre lines
• must be well characterized
– promoters can’t be leaky
• Andras Nagy’s database of Cre lines and other
knockout resources
http://www.mshri.on.ca/nagy/cre.htm
BioSci 145A lecture 12
page 10
©copyright
Bruce Blumberg 2000. All rights reserved
Regulated expression of introduced genes - Introduction
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Regulating gene expression at will in mammalian cells
has been a “Holy Grail” for molecular biologists.
– Constitutive, high-level expression of introduced
genes is not enough, fine tuning is essential
– genes must be repressible or inducible at will,
particularly those that are growth inhibitory or toxic
• apoptosis cascade.
– Levels of gene expression need to be monitored
during discrete time periods to understand regulatory
systems, such as signal transduction
• cultured cells
• animals
– cells that stably express deleterious proteins or
cytokines my be lost or phenotypes altered during
culture
Critical requirements
– Gene therapy requires tightly regulated expression
• modulated appropriately, not leaky
– time, place
• toxic levels of gene expression must be avoided
– high selectivity, shouldn’t interfere with other genes
– non-toxic inducer
• stability vs lability is relevant for experiments
– should work in many tissues
• blood brain barrier is an important obstacle
BioSci 145A lecture 12
page 11
©copyright
Bruce Blumberg 2000. All rights reserved
How is gene expression regulated?
BioSci 145A lecture 12
page 12
©copyright
Bruce Blumberg 2000. All rights reserved
Regulated expression - ecdysone
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Background
– No et al (1996) PNAS 93, 3346-3351
– 20-OH ecdysone is a steroid hormone that controls
metamorphosis in invertebrates
• family of hormones called ecdysteroids
– regulates transcription by interacting with a specific
cellular receptor, the ecdysone receptor
– functional ecdysone receptor is a heterodimer of two
different but related proteins, ecr and usp
(ultraspiracle)
• both partners of the heterodimer are required for
ligand binding and transcriptional activation
properties of the system
– ecdysone is not present in vertebrates and has no
detectable effects in rodents
• human effects?
– Activators are lipophilic molecules that can penetrate
most tissues, including brain
• muristerone A
• ponasterone A
– rapidly metabolized by cytochrome p450s
– not stored
– requires multiple components, RXR, EcR, EcRE
target gene construct.
BioSci 145A lecture 12
page 13
©copyright
Bruce Blumberg 2000. All rights reserved
Regulated expression - ecdysone (contd)
OH
20-OH ecdysone
OH
OH
OH
OH
O
cholesterol
OH
BioSci 145A lecture 12
page 14
©copyright
Bruce Blumberg 2000. All rights reserved
Regulated expression - ecdysone (contd)
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applications
– in vitro regulation of transfected genes
• muristerone A is not readily available in quantity
• other inducers are not synthetic, expensive
– regulating targeted gene disruption in ES cells and
embryos
advantages
– commercially available (InVitrogen, Stratagene)
– may have no deleterious effects in mammalian cells
– could work in transgenic animals if activators were
affordable and widely available
disadvantages
– requires multiple constructs/cell
– expense and unavailability of ligands
– little literature or experience
– questionable utility for gene therapy
– requires high concentration of ligand (~M)
caveats
– works fairly well in cell culture
– figures in paper are misleading, doesn’t work as well
as claimed vs tetracycline system
• nuances of reporter construction.
BioSci 145A lecture 12
page 15
©copyright
Bruce Blumberg 2000. All rights reserved
Regulated expression of introduced genes - tetracycline
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Background
– Gossen and Bujard (1992) PNAS 89, 5547-5551 is
the original publication
– based on the E. coli tetracycline (tc) resistance
operon derived from Tn10.
• tetO - tetracycline operator
• tetR - tetracycline repressor protein.
– In the absence of tc, the wild-type protein
binds to tetO and represses transcription
– in the presence of tc, the repressor is
dissociated and repression is abrogated
– many fusion proteins and other mutations have been
engineered into the system to obtain desirable
transcriptional effects
properties of the original system (called std tet)
– Clontech - Tet-OFF is commercial product
– tetR is fused to VP16, strong transcriptional activator
from herpes simplex virus under the control of a
strong promoter
– tetO is placed adjacent to a minimal promoter, eg
CMV.
• Choice of minimal promoter has profound
effects on basal activity!
• Main difference between ecdysone system and
tet from the No et al paper is the use of different
minimal promoters, tk vs MTV
BioSci 145A lecture 12
page 16
©copyright
Bruce Blumberg 2000. All rights reserved
Regulated expression - tetracycline (contd)
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Properties (contd)
– the VP16-TetR fusion protein constitutively activates
transcription from promoters containing tetO in the
absence of tc or doxycycline (dox)
– in the presence of tc or dox, the repressor dissociates
from tetO and activation is lost.
– Typical amount of dox required for full activity is in
the ng/ml range, this is ~2 nM
Applications
– primarily used in cell culture, difficult to ensure a
continuous supply of tc or dox in embryos
– some literature on the use of this system in embryos
BioSci 145A lecture 12
page 17
©copyright
Bruce Blumberg 2000. All rights reserved
Regulated expression - tetracycline (contd)
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Caveats and pitfalls
– for best results, stable cell lines should be used.
• Viral vectors have recently simplified process
– effector plasmid must be in large excess to response
plasmid in transient transfections
– bovine serum may contain tetracycline or its relatives
advantages
– target gene expression in the absence of inducer
• may work better for some experiments,
occasionally turning a gene off
disadvantages
– may be difficult to completely abrogate expression of
target gene in transient transfections
• unpredictable inheritance of plasmids influences
• high intracellular concentrations of VP16-tetR
are required to ensure full promoter occupancy.
– may need to use small amounts of dox to
titrate toxic effects
– considerable optimization is required for success
– cell type specific differences in behavior are not
uncommon
– time lag for effects of tc or dox addition or removal
• 1/2 life of mRNA or protein
• clearance of drug
BioSci 145A lecture 12
page 18
©copyright
Bruce Blumberg 2000. All rights reserved
Regulated expression - reverse tetracycline
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Background
– Gossen et al (1995) Science 268, 1766-1769
– designed to behave like a more standard inducible
system to comfort some molecular biologists
• addition of inducer activates transcription
properties of the system
– mutated tetR such that binding of dox induces DNA
binding rather than abrogating it, rtetR.
– VP16-rtetR fusion is then an activator only in the
presence of dox (tc doesn’t work well)
applications
– appears to be more amenable to precise regulation
than std tet
– commonly used in transgenic mice
Caveats and pitfalls
– for best results, stable cell lines should be used.
• Viral vectors have recently simplified process
– minimal promoter selection CRITICAL for success
– bovine serum may contain tetracycline or its relatives
BioSci 145A lecture 12
page 19
©copyright
Bruce Blumberg 2000. All rights reserved
Regulated expression - reverse tetracycline (contd)
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advantages
– inducer only required to activate gene expression
• conceptually and practically easier
– no requirement for high levels of VP16-rtetR protein
as with std tet.
• Better for transient transfection than std tet
disadvantages
– somewhat leaky, basal expression can be problematic
• choice of minimal promoter
– much higher levels of dox required than for std tet toxicity is problematic
BioSci 145A lecture 12
page 20
©copyright
Bruce Blumberg 2000. All rights reserved
Regulated expression - hybrid tetracycline systems
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Background
– references
• Kringstein et al (1998) PNAS 95, 13670-75
• Baron et al (1999) PNAS 96, 1013-1018
• Blau and Rossi (1999) PNAS 96, 797-799
– utilizes highly engineered tet and reverse tet proteins
to get specific effects
properties of the system
– what happens when one puts proteins into the cell that
respond differently to the same effector compound?
• If they can dimerize with each other
• can not dimerize with each other
• or if they do not dimerize and bind to different
and non-overlapping operator sequences
– different function, can heterodimerize
• in this case, a fair number will make unproductive
heterodimers and interfere with desired effect
• this will also be problematic even if the two
dimers have been engineered to bind different
targets
• so for maximum effect, we must prevent
heterodimerization between effectors that
– can bind to different sequences
– have different functions (activator vs
repressor
BioSci 145A lecture 12
page 21
©copyright
Bruce Blumberg 2000. All rights reserved
Regulated expression - hybrid tetracycline (contd)
Heterodimers possible
same DNA targets
different DNA targets
Heterodimers not possible
functional discrimination
works
– Opposite function - same DNA target
• use pure tetR and VP16-rtetR
• at low dox, the repressor will dominate
• as dox increases, the repressor will dissociate and
VP16-rtetR will activate
• increases dynamic range of activation ~105 fold
– increases sensitivity as well
BioSci 145A lecture 12
page 22
©copyright
Bruce Blumberg 2000. All rights reserved
Regulated expression - hybrid tetracycline (contd)
– Opposite function - different DNA target
• use std tet with one type of tetO to regulate gene
A
• and rev tet with another type of tetO to regulate
gene B
• in the absence of dox, gene A will be activated
while B will be silent
• in the presence of high dox, gene A will be
inactivated and gene B will be activated
BioSci 145A lecture 12
page 23
©copyright
Bruce Blumberg 2000. All rights reserved
Regulated expression - hybrid tetracycline (contd)
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Applications
– activator and repressor
• very sensitive regulation of responsive gene
expression
• tightly regulated expression over 5 logs
• can readily measure effects of subtle changes in
gene expression
– how much change in expression is required
to get effects?
– Do effects differ at different levels of
expression?
– Activator/repressor two targets
• can create “conditional mutants” that have one
activity at low levels of effector substance and
another at high levels
• can regulate two different genes or two alleles of
a single gene
– mutually exclusive expression
– or expression of neither
• can repeatedly switch between two states and
observe effects at high resolution
• possible to perturb intracellular equilibria in
small increments and follow the effects on
phenotype
BioSci 145A lecture 12
page 24
©copyright
Bruce Blumberg 2000. All rights reserved
Regulated expression - hybrid tetracycline (contd)
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Caveats and pitfalls
– best done with stable cell lines
advantages
– much more versatile than ecdysone
– possible to fine tune expression of a single gene, or
two genes with unprecendented resolution
– can make conditional mutants without genetics
• eg in model systems not amenable to genetics
such as Xenopus or chicken
– can make conditional replacements in vivo
• knock the repressible tetO into an endogenous
gene
• introduce a transgene under the control of the
activatable tetO
• breed these mice with a line expressing the dual
tet repressors
• increasing dox will inactivate the endogenous
gene and activate the transgene
disadvantages
– technically demanding
– multiple steps required
– may not work as well as predicted due to complexity
– virus-based systems may not work in ES cells
– Clontech’s TRE-effector plasmids have high
background - need to be remade
BioSci 145A lecture 12
page 25
©copyright
Bruce Blumberg 2000. All rights reserved
Regulated gene expression - summary
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what system do you need to use?
– Transgenic animals?
– Gene therapy?
– Cell culture?
How much tolerance is there for modulation of non target
genes?
– Eg glucocorticoids, estrogens, progestins and
thyroid hormones are very important physiologically
and cannot be modulated without collateral effects in
adults
• however, these are very good in the early
embryo of model organisms
How stringent must the regulation be?
– The more stringent the requirements for control the
greater the likelihood that complex techniques will
be required
• eg hybrid tetracycline
Is there a need for regulating multiple genes?
– Hybrid tet is the only way to go
What are the commercial implications?
– Licenses may be required for various technologies
• tet is controlled by BASF
• ecdysone by the Salk Institute
– depending on the license agreement, one technique
might be preferred (eg ecdysone)
BioSci 145A lecture 12
page 26
©copyright
Bruce Blumberg 2000. All rights reserved
Gene transfer technology - implications
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Genetics and reverse genetics
– gene transfer and selection technology speeds up
genetic analysis by orders of magnitude
– virtually all conceivable experiments are now
possible
• all questions are askable
– much more straightforward to understand gene
function using knockouts and transgenics
• gene sequences are coming at an unprecedented
rate from the genome projects
• Knockouts and transgenics remain very
expensive to practice
– other yet undiscovered technologies will be
required to understand gene function.
Clinical genetics
– Molecular diagnostics are becoming very widespread
as genes are matched with diseases
• huge growth area for the future
• big pharma is dumping billions into diagnostics
– room for great benefit and widespread abuse
• diagnostics will enable early identification and
treatment of diseases
• but insurance companies will want access to
these data to maximize profits
BioSci 145A lecture 12
page 27
©copyright
Bruce Blumberg 2000. All rights reserved
Gene transfer technology - implications (contd)
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gene therapy
– new viral vector technology is making this a reality
• now possible to get efficient transfer and
reasonable regulation
– long lag time from laboratory to clinic, still working
with old technology in many cases
protein engineering
– not as widely appreciated as more glamorous
techniques such as gene therapy and transgenic crops
– better drugs, eg more stable insulin, TPA for heart
attacks and strokes, etc.
– more efficient enzymes (e.g. subtilisin in detergents)
– safe and effective vaccines
• just produce antigenic proteins rather than using
inactivated or attenuated organisms to reduce
undesirable side effects
metabolite engineering
– enhanced microbial synthesis of valuable products
• eg indigo (jeans)
• vitamin C
– generation of entirely new small molecules
• transfer of antibiotic producing genes to related
species yields new antibiotics (badly needed)
– reduction of undesirable side reactions
• faster more efficient production of beer
BioSci 145A lecture 12
page 28
©copyright
Bruce Blumberg 2000. All rights reserved
Gene transfer technology - implications (contd)
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transgenic food
– gene transfer techniques have allowed the creation of
desirable mutations into animals and crops of
commercial value
• disease resistance (various viruses)
• pest resistance (Bt cotton)
• pesticide resistance
• herbicide and fungicide resistance
• growth hormone and milk production
– effective but necessary?
– negative implications
• pesticide and herbicide resistance lead to much
higher use of toxic compounds
• results are not predictable due to small datasets
• at least one herbicide (bromoxynil) for which
resistance was engineered has since been banned
plants as producers of specialty chemicals
– still very underutilized since plant technology yet
lags behind techniques in animals
– great interest in using plants as factories to produce
materials more cheaply and efficiently
• especially replacements for petrochemicals
– plants and herbs are the original source of many
pharmaceutical products hence it remains possible to
engineer them to overproduce desirable substances
BioSci 145A lecture 12
page 29
©copyright
Bruce Blumberg 2000. All rights reserved