Transcript No Slide Title - Bruce Blumberg

BioSci 145B Lecture #8 5/25/2004
• 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 8
page 1
©copyright
Bruce Blumberg 2004. All rights reserved
Methods of profiling gene expression – large scale
• Global analysis of RNA expression – identifying all expressed sequences,
a.k.a. the transcriptome
– Array – micro or macro
– Sequence sampling
– SAGE – serial analysis of gene expression
– Massively parallel signature sequencing
• DNA microarray analysis is now totally dominant method
– Two basic flavors
• Spotted (spot DNA onto support)
– cDNA microarrays
– Oligonucleotide arrays
– Moderately expensive
• Synthesized (use photolithography to synthesize oligos onto silicon or
other suitable support
– Affymetrix Gene Chips dominate
– VERY expensive
– Both are in wide use and suitable for whole genome analysis
BioSci 145B lecture 8
page 2
©copyright
Bruce Blumberg 2004. All rights reserved
Types of microarray fabrication
• Photolithography
– Uses light to covalently attach
the DNA
– Availability: GeneChip
(Affymetrix)
• Mechanical spotting
– Spotting pins to transport DNA
– Availability: academic facilities
& Vendors
• Ink jet (piezoelectric) printing
– Uses electric current to
dispense DNA
– Availability: Agilent
BioSci 145B lecture 8
page 3
©copyright
Bruce Blumberg 2004. All rights reserved
Affymetrix GeneChips
• High density arrays are synthesized directly on support
– 4 masks required per cycle -> 100 masks per chip (25-mers)
– Pentium IV requires about 30 masks
– G.P. Li in Engineering directs a UCI facility that
can make just about anything using photolithography
BioSci 145B lecture 8
page 4
©copyright
Bruce Blumberg 2004. All rights reserved
Affymetrix GeneChips
Streptavidin/phycoerythrin
BioSci 145B lecture 8
page 5
©copyright
Bruce Blumberg 2004. All rights reserved
Affymetrix GeneChips
– Each gene is represented by a series of oligonucleotide pairs
• One perfect match
• One with a single mismatch
– hybridization to perfect match but not mismatch is considered to be real
– Gene is considered “detected” if
> ½ of oligo pairs are positive
– Number of pairs depends on
organism and how well
characterized array behavior is
• Xenopus uses 16 pairs
BioSci 145B lecture 8
page 6
©copyright
Bruce Blumberg 2004. All rights reserved
Affymetrix GeneChips
• Result is in single color
– Need two chips – control and experimental for each condition
• Advantages
– Commercially available
– Standardized
• Disadvantages
– About $1000 to buy, probe and
process each chip at UC
prices.
• My Japanese collaborators
pay > $3,000/chip
– May not be available for your
organism of interest
– No ability to compare probes
directly on the same chip
• Must rely on technology
BioSci 145B lecture 8
page 7
©copyright
Bruce Blumberg 2004. All rights reserved
Spotted arrays
• Source material is prepared
– cDNAs are PCR amplified OR
– Oligonucleotides synthesized
• Spotted onto treated glass slides
• RNA prepared from 2 sources
– Test and control
• Labeled cDNA probes are prepared
from Ranks by reverse transcription
– Incorporate label directly
– Or incorporate modified NTP
and label later
– Or chemically label mRNA directly
• Hybridize, wash, scan slide
• Express as ratio of one channel to
other after processing
BioSci 145B lecture 8
page 8
©copyright
Bruce Blumberg 2004. All rights reserved
DNA microarray types
• Stanford type
microarrayer
– http://cmgm.stanford
.edu/pbrown/mguide/
index.html
• Printing method
– Reminiscent of
fountain pen
BioSci 145B lecture 8
page 9
©copyright
Bruce Blumberg 2004. All rights reserved
Strategy to identify RAR target genes
Agonist - TTNPB
Antgonist - AGN193109
Harvest st 18
Poly A+ RNA
Poly A+ RNA
Amino-allyl labeled
1st strand cDNA
Amino-allyl labeled
1st strand cDNA
Alexa Fluor
555 (cy3)
Alexa Fluor
647 (cy5)
Alexa Fluor
555 (cy3)
Alexa Fluor
647 (cy5)
Probe microarrays
upregulated
BioSci 145B lecture 8
page 10
©copyright
downregulated
Bruce Blumberg 2004. All rights reserved
DNA microarray
• Statistical analysis of output – VERY IMPORTANT!
• Replicates are very important
• Preprocessing of data is needed
– To remove spurious signals
BioSci 145B lecture 8
page 11
©copyright
Bruce Blumberg 2004. All rights reserved
DNA microarray
• Advantages
– Custom arrays possible and
affordable
– Ratio of fluorescence is robust
and reproducible
• Disadvantages
– Availability of chips
– Expense of production on your own
– Technical details in preparation
• If you want to see a microarrayer, drop by my
lab and I will show you several types
BioSci 145B lecture 8
page 12
©copyright
Bruce Blumberg 2004. All rights reserved
DNA microarrays
• What are they good for?
– Identifying genes expressed in one condition vs another
• One tissue vs another (heart vs liver)
• Tissue vs tumor (liver vs hepatocarcinoma)
• In response to a treatment (e.g., RA)
• In response to disease (after viral infection)
– Building expression profiles
• Tissues
• Cancers
• Developmental stages
• Expressed genes on chromosomes
– Identifying organisms
• Array can identify which types of animals are present in a mix
• http://www.affymetrix.com/corporate/media/genechip_essentials/f
oodexpert/FoodExpert_ID_Array.affx
BioSci 145B lecture 8
page 13
©copyright
Bruce Blumberg 2004. All rights reserved
DNA microarrays
• What are they good for? (contd)
– Response of animal to drugs or chemicals
• Toxicogenomics http://www.niehs.nih.gov/nct/home.htm
– Diagnostics
• SNP analysis to identify disease loci
• Specific testing for known diseases
BioSci 145B lecture 8
page 14
©copyright
Bruce Blumberg 2004. All rights reserved
Global profiling of protein expression
• Proteomics is broad name given to study of the proteome
– Proteome -> a cell or organism’s complement of expressed proteins
• Methods
– 2-D gel electrophoresis
– Mass spectrometry of various sorts
• All mass spec requires that molecules “fly” and measures
mass/charge (m/z) ratio
• MALDI-TOF
– Matrix assisted laser desorption ionization – time of flight
– Laser causes matrix to vaporize and molecules to fly, charge is
applied and time molecule takes to fly to detector measured
along with m/z
• ESI
– electrospray ionization – molecules are sprayed, ionized and
detected
• MS-MS
– Tandem mass spec – has two mass analyzers - first detector
shunts selected molecule to second – used for sequencing and
structure analysis
BioSci 145B lecture 8
page 15
©copyright
Bruce Blumberg 2004. All rights reserved
Global profiling of protein expression (contd)
• 2-D electrophoresis
– Ironically, this is the oldest method for “proteomics”
– First dimension is isoelectric focusing
• Set up a pH gradient in tube, apply proteins and electrophorese
• each protein goes to its isoelectric point and stops
– Second dimension is SDS-PAGE – proteins migrate according to size
• Run at 90º to first dimension
– Current technology is to cut out spots and id by mass spec
• Mass spec resurrected 2-D electrophoresis
–
Steep pH gradient
shallow pH gradient
BioSci 145B lecture 8
page 16
©copyright
Bruce Blumberg 2004. All rights reserved
Global profiling of protein expression (contd)
• 2-D electrophoresis (contd)
– Good points
• Straightforward separation
• Can get good resolution with good isoelectric focusing gels
– Downside
• Protein may not be detectable as well-resolved spots that can be
excised and characterized
– Co-migrate
– Abundance
• Variation from experiment to experiment
– Spot position on gel is very sensitive to small changes in pH
BioSci 145B lecture 8
page 17
©copyright
Bruce Blumberg 2004. All rights reserved
Global profiling of protein expression (contd)
• Mass spectrometric methods
– MudPIT is most useful for large scale protein profiling
• Multidimensional protein identification technology
– Separate proteins by microcapillary liquid chromatography
– Characterize and identify proteins by ms-ms
– Lan Huang is local expert on protein profiling by mass spectrometry
• http://www.ucihs.uci.edu/pandb/faculty/Huang.htm
BioSci 145B lecture 8
page 18
©copyright
Bruce Blumberg 2004. All rights reserved
Global profiling of protein expression (contd)
• Strategies for high-throughput,
high resolution protein
identification and analysis
– Equipment is very expensive
but possibilities are limitless
– Can match proteins with
database sequences OR
– Can sequence proteins de
novo
• Computationally
intensive
BioSci 145B lecture 8
page 19
©copyright
Bruce Blumberg 2004. All rights reserved
Global profiling of protein expression (contd)
• Protein arrays now available
– Immobilized proteins
• Spot proteins on slides and ask what interacts with particular ones
• Luis Villareal runs a facility here that intends to produce all possible
proteins for array generation
– Antibody arrays
• Antibodies spotted on arrays – test for presence of particular proteins
in probe
• Micro-ELISA or RIA
– Antigen arrays
• Known antigens spotted – tests for presence of antibodies in sample
BioSci 145B lecture 8
page 20
©copyright
Bruce Blumberg 2004. 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 followed by 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
• Because we have identified many genes from sequencing with no
known functions, or even hints about function
– 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 145B lecture 8
page 21
©copyright
Bruce Blumberg 2004. 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 145B lecture 8
page 22
©copyright
Bruce Blumberg 2004. 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 recent review is Müller (1999) Mechanisms of Development
82, 3-21.
BioSci 145B lecture 8
page 23
©copyright
Bruce Blumberg 2004. All rights reserved
Gene targeting (contd)
• 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 stem cell phenotype
• 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 145B lecture 8
page 24
©copyright
Bruce Blumberg 2004. All rights reserved
Gene targeting (contd)
• 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 145B lecture 8
page 25
©copyright
Bruce Blumberg 2004. All rights reserved
Gene Targeting (contd)
• Targeting vector
• Electroporate into ES
cells
• Recombination
• Selection
• identification
BioSci 145B lecture 8
page 26
©copyright
Bruce Blumberg 2004. 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 is best
• locus targeted. This may
result from differences in
chromatin structure and
accessibility
– Expand ES cell colonies
BioSci 145B lecture 8
page 27
©copyright
Bruce Blumberg 2004. All rights reserved
Gene targeting (contd)
– 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 145B lecture 8
page 28
©copyright
Bruce Blumberg 2004. 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 145B lecture 8
page 29
©copyright
Bruce Blumberg 2004. 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)
• 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 8
page 30
©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 8
page 31
©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 8
page 32
©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 8
page 33
©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 8
page 34
©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 8
page 35
©copyright
Bruce Blumberg 2004. All rights reserved