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BioSci D145 Lecture #8
• Bruce Blumberg ([email protected])
– 4103 Nat Sci 2 - office hours Tu, Th 3:30-5:00 (or by appointment)
– phone 824-8573
• TA – Riann Egusquiza ([email protected])
– 4351 Nat Sci 2– office hours M 1-3
– Phone 824-6873
• Updated lectures will be posted on web pages after lecture
– http://blumberg-lab.bio.uci.edu/biod145-w2017
• Term papers due Friday, March 10 by 12
midnight (23:59.59) (-1 point for each day late)
BioSci D145 lecture 8
page 1
©copyright
Bruce Blumberg 2004-2016. All rights reserved
Term paper requirements and scoring
• Actual paper – 5 pages single spaced 1” margins (references not included).
• PLEASE DO NOT WRITE AN ABSTRACT
• Specific aims – 2 points (this should be about 3/4 to one page)
– Write a paragraph introducing the topic, state why it is important and
what are the gaps in knowledge that you will address.
– State a hypothesis to be tested
– Enumerate 2-3 specific aims in the form of questions that test your
hypothesis. Use 1-2 sentences to state how you will answer the question
– Finish with a paragraph describing what is innovative about your work
and what impact it will have. It is VERY IMPORTANT to state
• the human health relevance of your research (if you are doing
something biomedical) or
• the broader impacts on advancing the frontiers of knowledge (for
something that is not relevant to human health).
• This is a critically important part of a grant application. You have to
convince the reviewer that your work is important and worth funding.
BioSci D145 lecture 1
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©copyright
Bruce Blumberg 2004-2016. All rights reserved
Term paper requirements and scoring
• Background and Significance – 3 points (about 1.5-2.5 pages)
– Briefly summarize what is known about the problem.
• Not a comprehensive review, just a summary of the important points.
– Succinctly state what is not known and why it is important that this
research be done
• Address knowledge gaps
• Are you addressing something controversial?
– talk about the controversy and why your work will address it
directly.
– In about one paragraph, state what is important about your proposed
research and why will accomplishing it benefit the research community
and world at large.
• i.e., what is the potential impact if you are successful
• Don’t repeat what was said in specific aims exactly but obviously
they should be related.
• http://blumberg-lab.bio.uci.edu/biod145-w2017/example_grant.pdf
BioSci D145 lecture 1
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©copyright
Bruce Blumberg 2004-2016. All rights reserved
Term paper requirements and scoring
• Research plan – 4 points (about 2.5-3 pages)
– In a short paragraph, state what you will do and why it is important. (I
know it seems repetitive by now, but reviewers are busy and will be
skimming your grant. You need to hit them over the head a few times
before they will get your point).
– Restate each specific aim from the Specific aims section (one by one)
– describe what you will do to address the aim
• Break into subaims as appropriate
• State the hypothesis to be tested in each
• Explain the rationale
• Describe briefly what approach you will take
• Discuss what you expect to find
• Point out any possible problems and alternative approaches
– I am mostly concerned with your hypothesis and rationale here.
– Not an all-encompassing proposal – 4-5 years by a small team (e.g., your
PhD thesis research)
BioSci D145 lecture 1
page 4
©copyright
Bruce Blumberg 2004-2016. All rights reserved
Functional Genomics - The challenge: Many new genes of unknown function
• Where/when are they expressed?
– Known genes (e.g. from genome projects)
• Gene chips (Affymetrix)
• Microarrays (Oligo, cDNA, protein)
– Novel genes
• Differential display
• Expression profiling (3 papers this week)
– SAGE and related approaches
– Massively parallel sequencing
– Which genes regulate what other genes?
• ChIP (chromatin immunoprecipitation)
• What is the phenotype of loss-of-function?
– Gene targeting by homologous recombination
– Genome wide siRNA
– CRISPR-Cas9
BioSci D145 lecture 8
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©copyright
Bruce Blumberg 2004-2016. All rights reserved
Genome wide analysis of gene function
• Loss-of-function analysis is the most powerful way to identify gene function
– Direct link between genotype and phenotype
– Forward vs reverse genetics
• Forward genetics-> random mutagenesis, then phenotypic analysis
– Identity of gene involved not known at the start
• Reverse genetics -> associating functions with known genes
– Directed mutagenesis of individual genes, phenotypic analysis
• Reverse genetics is much more important than forward genetics in
post genomic era – why?
– Because we have identified many genes by sequencing with no
known functions, or even hints about function
– What are some useful approaches ?
• Mutagenesis (forward genetics)
• Systematically mutating each gene (required genome sequence)
• Random targeting with viruses or transposons, match genes later
– Can id new genes as well as known genes
• Generate phenocopies of mutant alleles
– RNAi (siRNA), morpholinos, virus induced gene silencing
BioSci D145 lecture 8
page 6
©copyright
Bruce Blumberg 2004-2016. All rights reserved
Construction of transgenic animals
• standard transgenesis
– Microinject DNA into a fertilized egg (mouse)
or embryo (Drosophila)
• some embryos undergo integration of
DNA into genome
• transgene goes to offspring in a few
– Why do these mice have stubby tails?
• Applications
– rescue of a mutation
– promoter analysis
• ID elements required for expression
• verify function of putative elements
– model for dominant forms of human diseases
– identify effects of misexpression
– Large scale mutagenesis
• Gene trap
• Enhancer trap
• RNAi
BioSci D145 lecture 8
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©copyright
Bruce Blumberg 2004-2016. All rights reserved
Gene targeting
• Transgenesis is mostly a gain-of-function technique
– Loss-of-function preferred for identifying gene function
• Targeted gene disruption is very desirable
– to understand function of newly identified genes
• e.g., from genome projects
• Or gene by gene
– 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 review is Müller (1999) Mechanisms of Development 82, 321.
BioSci D145 lecture 8
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©copyright
Bruce Blumberg 2004-2016. All rights reserved
Gene targeting (contd)
• enabling technology is embryonic
stem (ES) cells (or iPS 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 stem cell phenotype
• Also problematic for human ES lines
• 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
– ES cells depend critically on the culture conditions maintain an
uncommitted, undifferentiated state that allows germ line transmission.
BioSci D145 lecture 8
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©copyright
Bruce Blumberg 2004-2016. All rights reserved
Gene targeting (contd) stopped here 2015
• isolate genomic clones from
ES cell library
• Restriction map
– Especially exons/introns
• Make targeting construct
– Want ~5kb genomic regions
flanking targeted region
– Must disrupt essential exon
– Want no functional protein
– Verify in cell culture
– often useful to fuse reporter gene to the coding region of the protein
• gene expression can be readily monitored
– Insert dominant selectable marker within replacement region
– negative selection marker is located outside the region targeted to be
replaced
• Electroporate DNA into ES cells, select colonies resistant to positive selection
• Integration positive cells then subjected to negative selection
– homologous recombinants lose this marker
BioSci D145 lecture 8
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©copyright
Bruce Blumberg 2004-2016. All rights reserved
Gene Targeting (contd)
• Targeting vector
• Electroporate into ES
cells
• Recombination
• Selection
• identification
BioSci D145 lecture 8
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©copyright
Bruce Blumberg 2004-2016. All rights reserved
Gene targeting (contd)
• Technique (contd)
– homologous recombination is
verified by Southern blotting
– factors affecting targeting
frequency (success)
• 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 is best
• locus targeted. This may
result from differences in
chromatin structure and
accessibility
– Expand ES cell colonies
BioSci D145 lecture 8
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©copyright
Bruce Blumberg 2004-2016. All rights reserved
Gene targeting (contd)
– Transfer into blastocyst of recipient
– Implant into foster mothers (white or
black)
• Progeny will be mixed color
– Breed mixed color F1 mice with
homozygous white/black 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 D145 lecture 8
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©copyright
Bruce Blumberg 2004-2016. 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 (e.g. on opposite strand)
– interference from selection cassette
• strong promoters driving these may cause phenotypes
BioSci D145 lecture 8
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©copyright
Bruce Blumberg 2004-2016. All rights reserved
Gene targeting (contd)
• Applications
– creating loss-of-function alleles
– introducing subtle mutations
– chromosome engineering
– marking gene with reporter, enabling whole mount detection of
expression pattern (knock-in)
BioSci D145 lecture 8
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©copyright
Bruce Blumberg 2004-2016. All rights reserved
XhoI
KpnI
BamHI
EcoRI
EcoRI
1.3kbp
PstI
Short arm
7kbp
BamHI
Long arm
KpnI
EcoRI
BamHI
Example of a “knock-in” model
Wild type allele
E6 E7 E8
E9
loxP
E3 E4 E5
loxP
E2
Targeting vector
E3
Neo
PGK-DTA
I8
BGH-3’UTR
BamHI
BamHI
Human PXR LBD to C terminus
Targeted allele
E3
E2
Neo
NeoAL2
E9
SXR RC RV5
Southern Probe
Cre-recombined allele
E3
E2
BioSci D145 lecture 8
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©copyright
Bruce Blumberg 2006-2016 All rights reserved
E9
Gene targeting (contd)
• 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 (~30%)
– difficulties related to selection cassette
BioSci 145B lecture 6
page 17
©copyright
Bruce Blumberg 2006-2016. 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 D145 lecture 8
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©copyright
Bruce Blumberg 2006-2016 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
Chromosome
Target reversed
deleted for
target gene
e.g., Rosa26
BioSci D145 lecture 8
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©copyright
Bruce Blumberg 2006-2016 All rights reserved
Conditional gene targeting (contd)
• Strategy
– Make targeting construct
(minimum needed for grant)
– homologous recombination,
select for loss of DT-A
– 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 D145 lecture 8
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©copyright
3 possible outcomes
X
X
Bruce Blumberg 2006-2016 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://nagy.mshri.on.c
a/cre_new/index.php
BioSci D145 lecture 8
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©copyright
Bruce Blumberg 2006-2016 All rights reserved
Figure 5.25 Floxing mice
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/where not intended)
BioSci D145 lecture 8
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©copyright
Bruce Blumberg 2006-2016 All rights reserved
Other approaches to genome manipulation
• Transgenic and knockout technology is species dependent (doesn’t work in all
species – need ES cell equivalent). How else can we accomplish gene
disruption in a targeted way ?
– RNAi approaches (Luo paper this week)
– Nuclease based methods - introduce double-stranded breaks
• ZFN – zinc finger nucleases
• TALEN nucleases
• Meganuclease
– CRISPR/Cas9 – RNA guided nuclease (Gilbert, Liu papers this week)
• Meganucleases
– Based on naturally occurring restriction enzymes with extended DNA
binding specificity (relatively limited application)
• TALEN and ZFN nucleases
– Artificial fusion proteins combining an engineered DNA binding domain
fused to a nonspecific nuclease domain from FokI restriction enzyme
– ZNF – zinc finger repeats
– TALE – repeats
– Main limitation is the sequence specificity that can be engineered in.
BioSci D145 lecture 8
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©copyright
Bruce Blumberg 2006-2016 All rights reserved
CRISPR-Cas gene editing
• CRISPR = clustered, regularly interspaced, short palindromic repeat
Sander & Joung (2014) CRISPR-Cas
systems for editing, regulating and
targeting genomes, Nat Biotech
32: 347-355
Lander (2016) The Heroes of CRISPR
Cell 164, 18-28
– Cas9 – RNA-guided nuclease
– Functions as a bacterial immune system.
Foreign DNA is incorporated between
CRISPR repeat sequences, transcription
generates crRNA.
– Hybridizes to tracrRNA (also encoded by
CRISPR system), guides Cas9 nuclease to
the target
– Cas9 nuclease introduces double
stranded breaks into target
– Repair of this break can introduce
deletions frameshifts, etc.
BioSci D145 lecture 8
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©copyright
Bruce Blumberg 2006-2016 All rights reserved
CRISPR-Cas gene editing
• CRISPR/Cas9 (contd)
– For gene targeting, we would fuse
the target RNA to the tracrRNA and
introduce this into the target cell,
embryo, etc together with Cas9 nuclease
– Introduces ds breaks at target sequence
which may introduce desirable
mutations.
– Potential issues
• Can’t specify what happens after
ds break (deletion, frameshift,
insertion, etc
• Some question about specificity
of the crRNA introduced – may have
off-target effects
• Some sequence preferences of Cas9
nuclease may limit utility
BioSci D145 lecture 8
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©copyright
Bruce Blumberg 2006-2016 All rights reserved
CRISPR-Cas gene editing - applications
BioSci D145 lecture 8
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©copyright
Bruce Blumberg 2006-2016 All rights reserved
Generating phenocopies of mutant alleles
• How to inactivate endogenous genes in a targeted
but general way?
– 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
• variably in mammalian cells
• Poorly in Xenopus – Why?
Xenopus has an endogenous dsRNA helicase
BioSci D145 lecture 8
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©copyright
Bruce Blumberg 2006-2016 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 D145 lecture 9
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©copyright
Bruce Blumberg 2010. 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
• 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 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 D145 lecture 9
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©copyright
Bruce Blumberg 2010. All rights reserved
RNAi (contd)
• Parallels between siRNA and miRNA-directed RNAi
BioSci D145 lecture 9
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©copyright
Bruce Blumberg 2010. 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, loss of function ALWAYS partial
– can only generate phenotypes for a short time after introduction
BioSci D145 lecture 9
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©copyright
Bruce Blumberg 2010. All rights reserved
RNAi (contd)
• Ways to generate short silencing RNAs in vivo – need 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 (or
viral vector), let dicer
generate siRNAs
– d) produce imperfect
hairpin from pol II
promoter, let dicer
generate miRNAs that
direct gene silencing
BioSci D145 lecture 8
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©copyright
Bruce Blumberg 2006-2016 All rights reserved
RNAi (contd)
• RNAi for whole genome functional analysis
– First generate library of constructs that generates siRNA or stRNA
• Or synthesize a complete library of siRNAs
– 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
• IMPORTANT!
– RNAi is inherently transient unless you are expressing the siRNAs from a
stably integrated plasmid
– RNAi is a knock-down (not knock-out) method.
BioSci D145 lecture 9
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©copyright
Bruce Blumberg 2010. All rights reserved
Phenotypic rescue by RNAi – synthetic lethal and related approaches **
• How can we find other members of pathways we already know something
about?
– Or, how can we find drugs that act on a pathway to kill cells (e.g.,
cancer cells?)
– Synthetic lethal is one relatively new and promising approach
• 2 mutations are synthetic lethal if either single mutation is viable but
the double mutant is lethal
BioSci D145 lecture 8
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©copyright
Bruce Blumberg 2006-2016 All rights reserved
Phenotypic rescue by RNAi – synthetic lethal and related approaches
• How can we find other members of pathways we already know something
about?
– In cancer screening, what if we combine a mutation and
a drug to find a combination that increases the kill rate, or reveals a
phenotype that is similar to the total loss of function?
• Could find novel drug targets (pathways that kill cells in presence of
siRNA+drug (usually sublethal amount of drug)
• Can find genes that are targeted by drugs – pathway analysis
• Could find new biomarkers that are required for cell viability
following drug treatment
BioSci D145 lecture 8
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©copyright
Bruce Blumberg 2006-2016 All rights reserved
Mohr et al., 2010, Genomic Screening with RNAi: Results and Challenges.
Annual Review of Biochemistry, 29: 37-64
BioSci D145 lecture 8
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©copyright
Bruce Blumberg 2006-2016 All rights reserved