Download PPT - Bruce Blumberg

BioSci 145B Lecture #9 6/1/2004
• Bruce Blumberg
– 2113E McGaugh Hall - office hours Wed 12-1 PM (or by appointment)
– phone 824-8573
– [email protected]
• TA – Curtis Daly [email protected]
– 2113 McGaugh Hall, 924-6873, 3116
– Office hours Tuesday 11-12
• lectures will be posted on web pages after lecture
– http://eee.uci.edu/04s/05705/ - link only here
– http://blumberg-serv.bio.uci.edu/bio145b-sp2004
– http://blumberg.bio.uci.edu/bio145b-sp2004
• DON’T FORGET –
TERM PAPERS ARE DUE BY 5 PM on FRIDAY JUNE 4
BioSci 145B lecture 9
page 1
©copyright
Bruce Blumberg 2004. All rights reserved
Requirements for the term paper
• Goals
– Analytical thinking
– Improved writing
• Select a topic related to genomic or proteomic analysis of an interesting
problem
– Talk with me about your topic
• Write a short paper (~5 pages) in the style of a research grant describing how
you will attack this problem (example is posted).
– Specific aims – questions, hypotheses
– Background and significance
• What is known, what remains to be learned
• why should someone give you money to study this problem?
– Research plan – specific experiments to answer the questions posed in
specific aims
• Expected vs unexpected results
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)
– 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?
– Not really feasible to specifically target 20-30K genes
• Difficulty
• Expense
• Inability to target all possibile loci
– Some efforts to make mouse collection
• Lexicon Genetics has a collection
– 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
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 splie-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
• As in Golling paper we heard about on Thursday
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 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 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 doublestranded 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
• poorly in mammalian cells
• Poorly in Xenopus
BioSci 145B lecture 9
page 14
©copyright
Bruce Blumberg 2004. All rights reserved
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
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
– Use the same cellular complex as
siRNAs
– Perfect matches -> target cleavage
– Imperfect matches -> translational
repression of target
BioSci 145B lecture 9
page 16
©copyright
Bruce Blumberg 2004. All rights reserved
RNAi (contd)
• Parallels between siRNA and miRNA-directed RNAi
BioSci 145B lecture 9
page 17
©copyright
Bruce Blumberg 2004. All rights reserved
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
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 (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
O
O
N
BioSci 145B lecture 9
page 21
©copyright
Bruce Blumberg 2004. All rights reserved
Antisense methods to knock out gene function (contd)
Oligodeoxyribonucleotide
O
B
O
Morpholino Oligonucleotide
O
B = A, C, T, G
O
B
N
O
O P N
O P O
O
O
O
B
O
N
O
O P N
O P O
O
O
BioSci 145B lecture 9
page 22
©copyright
Bruce Blumberg 2004. All rights reserved
B
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
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
– straight biochemistry
• Co-immunoprecipitation
• GST-pulldown
– Library based methods
• phage display
• Yeast two hybrid
• in vitro expression cloning
• You want to identify all proteins that interact with all other proteins
– 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
– purify cellular proteins that interact with your protein
• co-immunoprecipitation
• GST-pulldown
• affinity chromatography
• biochemical fractionation
– 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
BioSci 145B lecture 9
page 26
©copyright
Bruce Blumberg 2004. All rights reserved
Mapping protein-protein interactions (contd)
• GST (glutathione-Stransferase) pulldown assay
– Versatile and general
– Fuse protein of interest
to GST
– Incubate with cell or
tissue extracts
– Mix with glutathionesepharose 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
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
– Key factor required for success is
no activation domain in bait!
– approach
• transfect library into cells and either
select for survival or activation of
reporter gene
• purify and characterize positive clones
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