BioSci 145A lecture 12 page 1 © copyright Bruce Blumberg All rights reserved Lecture /14/2001 Transcription factors I Topics we will cover today –transgenic technology (contd from last time) Gene targeting conditional gene targeting –gene trapping –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

BioSci 145A lecture 12 page 2 © copyright Bruce Blumberg All rights reserved Gene targeting Targeted disruption of genes is very desirable, wave of the future –great to understand function of newly identified genes from genome projects produce a mutation and evaluate the requirements for your gene of interest –good to create mouse models for human diseases knockout the same gene disrupted in a human and may be able to understand disease better and develop efficacious treatments excellent recent review is Müller (1999) Mechanisms of Development 82, enabling technology is embryonic stem (ES) cells –these can be cultured but retain the ability to colonize the germ line –essential for transmission of engineered mutations –derived from inner cell mass of blastula stage embryos –grown on lethally irradiated “feeder” cells which help to mimic the in vivo condition essential for maintaining phenotype of cells

BioSci 145A lecture 12 page 3 © copyright Bruce Blumberg All rights reserved Gene targeting (contd) How to make ES cells ES cells are very touchy in culture –lose ability to colonize germ line with time –easily infected by “mysterious microorganisms” that inhibit ability to colonize germ line ko labs maintain separate hoods and incubators for ES cell work –overall, ES cells depend critically on the culture conditions to keep them in an uncommitted, undifferentiated state that allows colonization of the germ line.

BioSci 145A lecture 12 page 4 © copyright Bruce Blumberg All rights reserved Gene targeting (contd) technique –isolate genomic clones spanning the gene of interest from an ES cell library –construct a restriction map of the locus with particular emphasis on mapping the exons –create a targeting construct with large genomic regions flanking the region to be disrupted –an essential exon(s) must be disrupted such that no functional protein is produced from the gene this should be carefully tested in cell culture before mice are made –it is often useful to design the construct such that a reporter gene is fused to the coding region of the protein this enables gene expression to be readily monitored and often provides new information about the gene’s expression –dominant selectable marker is inserted within replacement region –negative selection marker is located outside the region targeted to be replaced –DNA is introduced by electroporation and colonies resistant to positive selection are selected. –Integration positive cells are subjected to negative selection to distinguish homologous recombinants homologous recombinants lose this marker

BioSci 145A lecture 12 page 5 © copyright Bruce Blumberg All rights reserved Gene targeting (contd) Targeting vector electroporate recombination positive selection with dominant selective marker negative selection to identify homologous recombinants

BioSci 145A lecture 12 page 6 © copyright Bruce Blumberg All rights reserved Gene targeting (contd)

BioSci 145A lecture 12 page 7 © copyright Bruce Blumberg All rights reserved Gene targeting (contd) Technique (contd) –homologous recombination is verified by Southern blotting –factors affecting targeting frequency length of homologous regions, more is better. –0.5 kb is minimum length for shortest arm isogenic DNA (ie, from the ES cells) used for targeting construct. Polymorphisms appear to matter locus targeted. This may result from differences in chromatin structure and accessibility –Expand ES cell colonies –Transfer into blastocyst of recipient –Implant into foster mothers (white) Progeny will be mixed color –Breed mixed color F1 mice with homozygous white mice –Black progeny derive from germ cells harboring the knockout Heterozygous for knockout –Breed these to establish lines and determine effects of homozygous mutations

BioSci 145A lecture 12 page 8 © copyright Bruce Blumberg All rights reserved Gene targeting (contd) problems and pitfalls –incomplete knockouts, ie, protein function is not lost but such weak alleles may be informative –alteration of expression of adjacent genes region removed may contain regulatory elements may remove unintended genes (opposite strand) –interference from selection cassette strong promoters driving these may cause phenotypes Applications –creating loss-of-function alleles –introducing subtle mutations –chromosome engineering –marking gene with reporter, enabling whole mount detection of expression pattern (knock-in) advantages –can generate a true loss-of-function alleles –precise control over integration sites –prescreening of ES cells for phenotypes possible –can also “knock in” genes disadvantages –not trivial to set up –may not be possible to study dominant lethal phenotypes –non-specific embryonic lethality is common –difficulties related to selection cassette

BioSci 145A lecture 12 page 9 © copyright Bruce Blumberg 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, 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 10 © copyright Bruce Blumberg All rights reserved Conditional gene targeting - contd

BioSci 145A lecture 12 page 11 © copyright Bruce Blumberg 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 12 © copyright Bruce Blumberg All rights reserved Conditional gene targeting (contd)

BioSci 145A lecture 12 page 13 © copyright Bruce Blumberg 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

BioSci 145A lecture 12 page 14 © copyright Bruce Blumberg 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 15 © copyright Bruce Blumberg 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 16 © copyright Bruce Blumberg 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 17 © copyright Bruce Blumberg 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. Uses –Insertional mutagenesis –Marking genes to id interesting ones –Gene cloning

BioSci 145A lecture 12 page 18 © copyright Bruce Blumberg 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 19 © copyright Bruce Blumberg All rights reserved How is gene expression regulated?

BioSci 145A lecture 12 page 20 © copyright Bruce Blumberg All rights reserved Regulated expression - ecdysone Background –No et al (1996) PNAS 93, –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 21 © copyright Bruce Blumberg All rights reserved Regulated expression - ecdysone (contd)

BioSci 145A lecture 12 page 22 © copyright Bruce Blumberg 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 23 © copyright Bruce Blumberg All rights reserved Regulated expression of introduced genes - tetracycline Background –Gossen and Bujard (1992) PNAS 89, 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 abolished –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 24 © copyright Bruce Blumberg 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 25 © copyright Bruce Blumberg 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 26 © copyright Bruce Blumberg All rights reserved Regulated expression - reverse tetracycline Background –Gossen et al (1995) Science 268, –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 27 © copyright Bruce Blumberg 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