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