lecture 12 - 2/15/2001 transcription factors iblumberg-lab.bio.uci.edu/past...

BioSci 145A lecture 12 page 1 ©copyright Bruce Blumberg 2000. All rights reserved

Lecture 12 - 2/15/2001 Transcription factors I

• 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

• I have posted lectures on the web site in 3 formats– PPT = Powerpoint (native format)– PDF = Adobe Acrobat Portable Document Format– RTF = Word outline (no pictures)


Gene trapping

• Observation is that various types of viruses andtransposable elements can be utilized to deliver DNA torandom 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 withenhancers removed from the LTRs

• may or may not disrupt expression


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

– β-gal was the most widely used reporter– GFP is now popular– β-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 anddoes 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 viralvectors



• Flavors of gene trapping (contd)– expressed gene trap (many variations possible)

• designed to fuse inserted reporter with codingsequences of endogenous gene

• goal is to cause loss of expression of endogenousgene and replace it with the transgene

• typically done in ES cells to generate a library ofinsertional mutagens

– also widely used in Drosophila and zebrafish• reporter expression duplicates the temporal and

spatial pattern of the endogenous gene• reporters used

– β-gal was the most widely used reporter– GFP is now popular– β-lactamase is seeing increasing use



– 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.


Conditional gene targeting

• 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 ofthe 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


Conditional gene targeting - contd


Conditional gene targeting (contd)

• 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



• 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 butmore 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 resourceshttp://www.mshri.on.ca/nagy/cre.htm


Regulated expression of introduced genes - Introduction

• Regulating gene expression at will in mammalian cellshas 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 monitoredduring discrete time periods to understand regulatorysystems, such as signal transduction

• cultured cells• animals

– cells that stably express deleterious proteins orcytokines my be lost or phenotypes altered duringculture

• 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


How is gene expression regulated?


Regulated expression - ecdysone

• 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 specificcellular receptor, the ecdysone receptor

– functional ecdysone receptor is a heterodimer of twodifferent but related proteins, ecr and usp(ultraspiracle)

• both partners of the heterodimer are required forligand 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 penetratemost tissues, including brain

• muristerone A• ponasterone A

– rapidly metabolized by cytochrome p450s– not stored– requires multiple components, RXR, EcR, EcRE

target gene construct.


Regulated expression - ecdysone (contd)

OH

OHOH

OH

O

OH

20-OH ecdysone

OH

cholesterol


Regulated expression - ecdysone (contd)

• 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 andembryos

• 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 (~µM)

• 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.


Regulated expression of introduced genes - tetracycline

• 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 proteinbinds to tetO and represses transcription

– in the presence of tc, the repressor isdissociated and repression is abrogated

– many fusion proteins and other mutations have beenengineered into the system to obtain desirabletranscriptional 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 astrong promoter

– tetO is placed adjacent to a minimal promoter, egCMV.

• Choice of minimal promoter has profoundeffects on basal activity!

• Main difference between ecdysone system andtet from the No et al paper is the use of differentminimal promoters, tk vs ∆MTV


Regulated expression - tetracycline (contd)

• Properties (contd)– the VP16-TetR fusion protein constitutively activates

transcription from promoters containing tetO in theabsence of tc or doxycycline (dox)

– in the presence of tc or dox, the repressor dissociatesfrom tetO and activation is lost.

– Typical amount of dox required for full activity is inthe 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


Regulated expression - tetracycline (contd)

• 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


Regulated expression - reverse tetracycline

• 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 regulationthan 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


Regulated expression - reverse tetracycline (contd)

• 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


Regulated expression - hybrid tetracycline systems

• 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 proteinsto 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 unproductiveheterodimers and interfere with desired effect

• this will also be problematic even if the twodimers have been engineered to bind differenttargets

• so for maximum effect, we must preventheterodimerization between effectors that

– can bind to different sequences– have different functions (activator vs

repressor


Regulated expression - hybrid tetracycline (contd)

– 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

Heterodimers possible

same DNA targets

different DNA targets

Heterodimers not possible

functional discriminationworks



– 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



• Applications– activator and repressor

• very sensitive regulation of responsive geneexpression

• 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 oneactivity at low levels of effector substance andanother at high levels

• can regulate two different genes or two alleles ofa single gene

– mutually exclusive expression– or expression of neither

• can repeatedly switch between two states andobserve effects at high resolution

• possible to perturb intracellular equilibria insmall increments and follow the effects onphenotype



• 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 geneticssuch 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


Regulated gene expression - summary

• what system do you need to use?– Transgenic animals?– Gene therapy?– Cell culture?

• How much tolerance is there for modulation of non targetgenes?– Eg glucocorticoids, estrogens, progestins and

thyroid hormones are very important physiologicallyand cannot be modulated without collateral effects inadults

• however, these are very good in the earlyembryo of model organisms

• How stringent must the regulation be?– The more stringent the requirements for control the

greater the likelihood that complex techniques willbe 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 techniquemight be preferred (eg ecdysone)


Gene transfer technology - implications

• 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 genefunction using knockouts and transgenics

• gene sequences are coming at an unprecedentedrate from the genome projects

• Knockouts and transgenics remain veryexpensive to practice

– other yet undiscovered technologies will berequired 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


Gene transfer technology - implications (contd)

• gene therapy– new viral vector technology is making this a reality

• now possible to get efficient transfer andreasonable regulation

– long lag time from laboratory to clinic, still workingwith 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 usinginactivated or attenuated organisms to reduceundesirable 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


Gene transfer technology - implications (contd)

• transgenic food– gene transfer techniques have allowed the creation of

desirable mutations into animals and crops ofcommercial 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 muchhigher 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 yetlags behind techniques in animals

– great interest in using plants as factories to producematerials more cheaply and efficiently

• especially replacements for petrochemicals– plants and herbs are the original source of many

pharmaceutical products hence it remains possible toengineer them to overproduce desirable substances

lecture 12 - 2/15/2001 transcription factors iblumberg-lab.bio.uci.edu/past...

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