DON’T FORGET – TERM PAPERS ARE DUE BY 5 PM on Thursday June 2 BioSci 145B Lecture #9 5/31/2005 Bruce Blumberg 2113E McGaugh Hall - office hours Wed 12-1 PM (or by appointment) phone 824-8573 blumberg@uci.edu TA – Suman Verma sverma@uci.edu 2113 McGaugh Hall, 924-6873, 3116 DON’T FORGET – TERM PAPERS ARE DUE BY 5 PM on Thursday June 2 Suman will not be here this week so please either drop the paper off to me or submit online using the drop box or E-mail it to me. The time stamp on your mail will determine whether it is on time or not BioSci 145B lecture 9 page 1 ©copyright Bruce Blumberg 2004. 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 Clearly a big obstacle for gene analysis How can this be overcome? Generate conditional knockouts either in particular tissues or after critical developmental windows pass Sauer (1998) Methods 14, 381-392. BioSci 145B lecture 9 page 2 ©copyright Bruce Blumberg 2004. All rights reserved

Conditional gene targeting - contd Approach recombinases perform site-specific excision between recognition sites FLP system from yeast 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 locus of crossover x in P1 (loxP) Recognized by Cre recombinase if loxP sites are directly repeated then deletions if inverted repeats then inversions result BioSci 145B lecture 9 page 3 ©copyright Bruce Blumberg 2004. All rights reserved

Conditional gene targeting (contd) Strategy Make targeting construct (minimum needed for grant) homologous recombination, transfect CRE, select for loss of tk Southern to select correct event Result called “floxed allele” inject into blastocysts, select chimeras establish lines cross with Cre expressing line and analyze function BioSci 145B lecture 9 page 4 ©copyright Bruce Blumberg 2004. All rights reserved

Conditional gene targeting (contd) Tissue- or stage-specific knockouts from crossing floxed mouse with specific Cre-expressing line 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 145B lecture 9 page 5 ©copyright Bruce Blumberg 2004. 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 regarding site and time of expression promoters can’t be leaky (expressed when not intended) BioSci 145B lecture 9 page 6 ©copyright Bruce Blumberg 2004. All rights reserved

Genome wide analysis of gene function How to mutate all genes in a given genome? Easy with microbial genomes – can mutate all yeast genes by homologous recombination Recombine in selectable marker Propagate strain and analyze phenotypes Homology region Unique oligonucleotide “barcodes” for PCR Selectable marker (antibiotic resistance) Target gene BioSci 145B lecture 9 page 7 ©copyright Bruce Blumberg 2004. All rights reserved

Genome wide analysis of gene function (contd) How about gene targeting in other organisms With more complex genomes and more genes? Huge undertaking to specifically target 30K+ genes in mammalian cells Difficulty Expense Inability to target all possible loci Some efforts to make mouse collection Lexicon Genetics has a collection of ES cells Drosophila collection as well Driving force behind these efforts is Genome annotation Drug target discovery (Lexicon) Functional analysis BioSci 145B lecture 9 page 8 ©copyright Bruce Blumberg 2004. All rights reserved

Genome wide analysis of gene function (contd) Main method for gene targeting in more complex organisms is random insertional mutagenesis Transposon mutagenesis Bacteria – Tn transposons Yeast - Ty transposons Drosophila - P- elements Vertebrates - Sleeping Beauty transposons Viral infection Typically retroviruses – host range selectivity is obstacle Gene or enhancer trapping – modified viruses or transposons BioSci 145B lecture 9 page 9 ©copyright Bruce Blumberg 2004. All rights reserved

Insertional mutagenesis - Gene trapping viruses and transposable elements can deliver DNA to random locations can disrupt gene function put inserted gene under the control of adjacent regulatory sequences BOTH enhancer trap is designed to bring inserted reporter gene under the control of local regulatory sequences 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 Hopkins zebrafish group used unmodified virus BioSci 145B lecture 9 page 10 ©copyright Bruce Blumberg 2004. All rights reserved

Insertional mutagenesis - Gene trapping (contd) enhancer trap (contd) expression only when integrate into an active transcription unit 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 advantages relatively simple to perform active promoters frequently targeted, perhaps due to open chromatin Disadvantages Inactive promoters probably not targeted insertional mutagenesis not the goal, and not frequent 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 145B lecture 9 page 11 ©copyright Bruce Blumberg 2004. All rights reserved

Insertional mutagenesis – Gene trapping (contd) expressed gene trap (many variations possible) goal -> ablate expression of endogenous gene, replace with transgene Make insertion construct – reporter, selection, polyA sites No promoter but has a splice-acceptor sequence 5’ of reporter Can only be expressed if spliced into an endogenous mRNA Transfer into embryonic cells, generate a library of insertional mutagens Mouse, Drospophila, zebrafish, frog reporter expression duplicates the temporal and spatial pattern of the endogenous gene BioSci 145B lecture 9 page 12 ©copyright Bruce Blumberg 2004. All rights reserved

Insertional mutagenesis - Gene trapping (contd) Expressed gene trapping (contd) advantages insertional mutagen gives information about expression patterns can be made homozygous 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 identify interesting ones Gene cloning BioSci 145B lecture 9 page 13 ©copyright Bruce Blumberg 2004. All rights reserved

Generating phenocopies of mutant alleles How to inactivate endogenous genes in a targeted but general way? Important new development is RNAi – RNA interference Observation is that introduction of double-stranded RNAs into cells lead to destruction of corresponding mRNA (if there is one) Principle is siRNA – small interfering RNAs These generate small single stranded RNAs that target mRNAs for destruction by RISC – RNA interference silencing complex First applied in C. elegans where it works extremely well Can introduce siRNA into cells even by feeding to the worms! Works very well in Drosophila variably in mammalian cells Poorly in Xenopus BioSci 145B lecture 9 page 14 ©copyright Bruce Blumberg 2004. All rights reserved

Dicer complex generates short duplexes from dsRNA in the cell RNAi (contd) Dicer complex generates short duplexes from dsRNA in the cell Important to have 2-nt overhangs siRNAs are generated from these fragments Antisense strand binds to mRNA and this recruits the RISC - RNAi silencing complex Complex leads to mRNA cleavage and destruction Two important reviews to read McManus and Sharp (2002) Nature Reviews Genetics 3, 737-747 Dykxhoorn et al. (2003) Nature Reviews, Molecular Cellular Biology 4, 457-467 BioSci 145B lecture 9 page 15 ©copyright Bruce Blumberg 2004. All rights reserved

Always form a hairpin structure with mismatches in stem RNAi (contd) Micro RNAs are small cellular RNAs that previously lacked any known function Always form a hairpin structure with mismatches in stem Turn out that micro RNAS direct gene silencing via translational repression (miRNAs) are mismatched duplexes that dicer processes into stRNAs (small temporal RNAs) Use same cellular complex as siRNAs Perfect matches -> target cleavage Imperfect matches -> translational repression of target Two important new papers Giraldez et al (2005) Science 308, 833-838 (microRNAs regulate brain morphogenesis) Lecellier et al (2005) Science 308, 557-560 (microRNA mediates antiviral defenses in human cells) BioSci 145B lecture 9 page 16 ©copyright Bruce Blumberg 2004. All rights reserved

Parallels between siRNA and miRNA-directed RNAi RNAi (contd) Parallels between siRNA and miRNA-directed RNAi BioSci 145B lecture 9 page 17 ©copyright Bruce Blumberg 2004. All rights reserved

Ways to generate short RNAs that silence gene expression in vitro RNAi (contd) Ways to generate short RNAs that silence gene expression in vitro a) chemical synthesis of siRNA, introduce into cell b) synthesize long dsRNA, use dicer to chop into siRNA c) introduce perfect duplex hairpin, dicer generates siRNA d) make miRNA based hairpin, dicer generates silencing RNA Introduce into cells or organism by microinjection, transfection, etc. Expression is transient can only generate phenotypes for a short time after introduction BioSci 145B lecture 9 page 18 ©copyright Bruce Blumberg 2004. All rights reserved

Ways to generate short silencing RNAs in vivo RNAi (contd) Ways to generate short silencing RNAs in vivo Continuing expression to generate stable phenotype a) produce long hairpin from pol II promoter, let dicer make siRNA b) produce two transcripts from pol III promoter, let anneal in cells c) produce a short hairpin from pol III promoter, let dicer generate siRNAs d) produce imperfect hairpin from pol II promoter, let dicer generate miRNAs that direct gene silencing BioSci 145B lecture 9 page 19 ©copyright Bruce Blumberg 2004. All rights reserved

RNAi for whole genome functional analysis RNAi (contd) RNAi for whole genome functional analysis First generate library of constructs that generates siRNA or stRNA Introduce these into cells, embyos (fly, frog, mouse) or animals (C. elegans, plants) For C. elegans, make the library in E. coli and simply feed bacteria to worms Must microinject or transfect with other animals Evaluate phenotypes BioSci 145B lecture 9 page 20 ©copyright Bruce Blumberg 2004. All rights reserved

Antisense methods to knock out gene function Antisense oligonucleotides can transiently target endogenous RNAs For destruction Many methods and oligo chemistries available Most are very sensitive to level of antisense oligo, these are degraded and rapidly muck up cellular nucleotide pools leading to toxicity For translational inhibition Morpholino oligos appear to work the best Morpholine sugar is substituted for deoxyribose Is not a substrate for cellular DNAses or RNAse H Base-pairs with RNA or DNA more avidly than standard DNA The oligo binds to the area near the ATG in the transcript and inhibits translation of the protein Deoxyribose morpholine BioSci 145B lecture 9 page 21 ©copyright Bruce Blumberg 2004. All rights reserved

Antisense methods to knock out gene function (contd) Oligodeoxyribonucleotide Morpholino Oligonucleotide B = A, C, T, G BioSci 145B lecture 9 page 22 ©copyright Bruce Blumberg 2004. All rights reserved

Most Molecules Function in Complexes Given a target, how can we identify interacting proteins? Complex members may be important new targets pharmacology toxicology Endocrine disrupter action High throughput, genome wide screen is preferred 20 years is too long BioSci 145B lecture 9 page 23 ©copyright Bruce Blumberg 2004. All rights reserved

Each protein interacts with average of 3 others How can we approach whole genome analysis of protein complex formation? Each protein interacts with average of 3 others Many are much more complex Two papers this Thursday and one next Thursday describe two different approaches to this problem. BioSci 145B lecture 9 page 24 ©copyright Bruce Blumberg 2004. All rights reserved

How to identify protein-protein interactions on a genome wide scale? You have one protein and want to identify proteins that interact with it You want to identify all proteins that interact with all other proteins straight biochemistry Co-immunoprecipitation GST-pulldown Library based methods phage display Yeast two hybrid in vitro expression cloning Proteomic analysis Protein microarrays Large scale two-hybrid BioSci 145B lecture 9 page 25 ©copyright Bruce Blumberg 2004. All rights reserved

Mapping protein-protein interactions biochemical approach – what are some ways to purify cellular proteins that interact with your protein pure protein(s) are microsequenced advantage functional approach stringency can be manipulated can identify multimeric proteins or complexes will work if you can purify proteins disadvantages much skill required low throughput considerable optimization required co-immunoprecipitation GST-pulldown affinity chromatography biochemical fractionation BioSci 145B lecture 9 page 26 ©copyright Bruce Blumberg 2004. All rights reserved

Mapping protein-protein interactions (contd) GST (glutathione-S-transferase) pulldown assay Versatile and general Fuse protein of interest to GST Incubate with cell or tissue extracts Mix with glutathione-sepharose beads Binds GST-fusion protein and anything bound to it Run SDS-PAGE Identify bands Co-Ip (immunoprecipitation) is identical except that antibody is used to pull down protein X BioSci 145B lecture 9 page 27 ©copyright Bruce Blumberg 2004. All rights reserved

Mapping protein-protein interactions (contd) scintillation proximity assay Target is bound to solid phase – bead or plate radioactive protein or ligand is added and allowed to reach equilibrium 35S, 125I, 3H work best radioactive decay is quenched in solution, only detected when in “proximity” of the solid phase, e.g. when bound to target applications ligand-receptor binding with 3H small molecules protein:protein interaction protein:DNA BioSci 145B lecture 9 page 28 ©copyright Bruce Blumberg 2004. All rights reserved

Mapping protein-protein interactions (contd) FRET - fluorescent resonance energy transfer based on the transfer of energy from one fluor to another that is not normally excited at that wavelength Many types of fluorescent moieties possible rare earth metals europium cryptate fluorescent proteins GFP and variants allophycocyanin Tryptophan residues in proteins application very commonly used for protein:protein interaction screening in industry FRET microscopy can be used to prove interactions between proteins within single cells Roger Tsien at UCSD is expert BioSci 145B lecture 9 page 29 ©copyright Bruce Blumberg 2004. All rights reserved

Mapping protein-protein interactions (contd) FRET (contd) advantages can be very sensitive may be inexpensive or not depending on materials non-radioactive equilibrium assay single cell protein:protein interactions possible time resolved assays possible disadvantage poor dynamic range - 2-3 fold difference full scale must prepare labeled proteins or ligands Not suitable for whole genome analysis tunable (or multiwavelength capable) fluorometer required (we have one here) BioSci 145B lecture 9 page 30 ©copyright Bruce Blumberg 2004. All rights reserved

Mapping protein-protein interactions (contd) Biacore (surface plasmon resonance) surface plasmon waves are excited at a metal/liquid interface Target bound to a thin metal foil and test sample flowed across it Foil is blasted by a laser from behind SPR alters reflected light intensity at a specific angle and wavelength Binding to target alters refractive index which is detected as change in SPR Change is proportional to change in mass and independent of composition of binding agent BioSci 145B lecture 9 page 31 ©copyright Bruce Blumberg 2004. All rights reserved

Mapping protein-protein interactions (contd) Biacore (contd) Advantages Can use any target Biological extracts possible Measure kinetics Small changes detectable with correct instrument 360 d ligand binding to 150 kd antibody Can use as purification and identification system Disadvantages Machine is expensive (we have two) “high throughput” very expensive Not trivial to optimize BioSci 145B lecture 9 page 32 ©copyright Bruce Blumberg 2004. All rights reserved

Library-based methods to map protein-protein interactions (contd) Phage display screening (a.k.a. panning) requires a library that expresses inserts as fusion proteins with a phage capsid protein most are M13 based some lambda phages used prepare target protein as affinity matrix or as radiolabeled probe test for interaction with library members if using affinity matrix you purify phages from a mixture if labeling protein one plates fusion protein library and probes with the protein called receptor panning based on similarity with panning for gold BioSci 145B lecture 9 page 33 ©copyright Bruce Blumberg 2004. All rights reserved

Library-based methods to map protein-protein interactions (contd) Phage display screening (a.k.a. panning) (contd) advantages stringency can be manipulated if the affinity matrix approach works the cloning could go rapidly disadvantages Fusion proteins bias the screen against full-length cDNAs Multiple attempts required to optimize binding Limited targets possible may not work for heterodimers unlikely to work for complexes panning can take many months for each screen Greg Weiss in Chemistry is local expert BioSci 145B lecture 9 page 34 ©copyright Bruce Blumberg 2004. All rights reserved

Mapping protein-protein interactions (contd) Two hybrid screening originally used in yeast, now other systems possible prepare bait - target protein fused to DBD (GAL4) usual stable cell line is commonly used prepare fusion protein library with an activation domain - prey What is the key factor required for success? approach transfect library into cells and either select for survival or activation of reporter gene purify and characterize positive clones no activation domain in bait! BioSci 145B lecture 9 page 35 ©copyright Bruce Blumberg 2004. All rights reserved

Mapping protein-protein interactions (contd) Two-hybrid screening (contd) Can be easily converted to genome wide searching by making haploid strains, each containing one candidate interactor Mate these and check for growth or expression of reporter gene Bait plasmid Prey plasmid If interact, reporter expressed and/or Yeast survive BioSci 145B lecture 9 page 36 ©copyright Bruce Blumberg 2004. All rights reserved

Mapping protein-protein interactions (contd) In vitro interaction screening - based on in vitro expression cloning (IVEC) transcribe and translate cDNAs in vitro into small pools of proteins (~100) test for their ability to interact with your protein of interest EMSA co-ip FRET SPA advantages functional approach smaller pools increase sensitivity diversity of targets proteins, complexes, nucleic acids, protein/nucleic acid complexes, small molecule drugs very fast disadvantages can’t detect heterodimers unless 1 partner known expensive consumables (but cheap salaries) Typical screen may cost $10-15K expense of automation BioSci 145B lecture 9 page 37 ©copyright Bruce Blumberg 2004. All rights reserved