1. 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
TA – Suman Verma
2113 McGaugh Hall, , 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
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 All rights reserved

2. 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,
BioSci 145B lecture 9 page 2 ©copyright Bruce Blumberg All rights reserved

3. 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 All rights reserved

4. 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 All rights reserved

5. 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
BioSci 145B lecture 9 page 5 ©copyright Bruce Blumberg All rights reserved

6. 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 All rights reserved

7. 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 All rights reserved

8. 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 All rights reserved

9. 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 All rights reserved

10. 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 All rights reserved

11. 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 All rights reserved

12. 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 All rights reserved

13. 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 All rights reserved

14. 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 All rights reserved

15. 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,
Dykxhoorn et al. (2003) Nature Reviews, Molecular Cellular Biology 4,
BioSci 145B lecture 9 page 15 ©copyright Bruce Blumberg All rights reserved

16. 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, (microRNAs regulate brain morphogenesis)
Lecellier et al (2005) Science 308, (microRNA mediates antiviral defenses in human cells)
BioSci 145B lecture 9 page 16 ©copyright Bruce Blumberg All rights reserved

17. 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 All rights reserved

18. 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 All rights reserved

19. 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 All rights reserved

20. 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 All rights reserved

21. 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 All rights reserved

22. 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 All rights reserved

23. 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 All rights reserved

24. 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 All rights reserved

25. 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 All rights reserved

26. 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 All rights reserved

27. 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 All rights reserved

28. 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 All rights reserved

29. 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 All rights reserved

30. 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 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 All rights reserved

31. 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 All rights reserved

32. 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 All rights reserved

33. 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 All rights reserved

34. 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 All rights reserved

35. 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 All rights reserved

36. 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 All rights reserved

37. 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 All rights reserved