BioSci 145B lecture 8 page 1 © copyright Bruce Blumberg 2004. All rights reserved BioSci 145B Lecture #8 5/25/2004 Bruce Blumberg –2113E McGaugh Hall -

BioSci 145B lecture 8 page 1 ©copyright Bruce Blumberg 2004. All rights reserved

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

mailto:[email protected]

http://eee.uci.edu/04s/05705/

http://blumberg-serv.bio.uci.edu/bio145b-sp2004

http://blumberg.bio.uci.edu/bio145b-sp2004


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


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


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



Streptavidin/phycoerythrin



– 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



• 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 UCprices.

• My Japanese collaboratorspay > $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


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


DNA microarray types

• Stanford type microarrayer– http://

cmgm.stanford.edu/pbrown/mguide/index.html

• Printing method – Reminiscent of

fountain pen


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 Fluor555 (cy3)




Probe microarrays

upregulated downregulated


DNA microarray

• Statistical analysis of output – VERY IMPORTANT!• Replicates are very important• Preprocessing of data is needed

– To remove spurious signals


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


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/foodexpert/FoodExpert_ID_Array.affx


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


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


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



• 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



• 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



• 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



• 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


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


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


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.


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.


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


Gene Targeting (contd)

• Targeting vector

• Electroporate into ES cells

• Recombination

• Selection

• identification



• 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



– 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



• 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



• 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


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.


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


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



– Tissue- or stage-specific knockouts from crossingfloxed 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



• 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)

Documents

BioSci 145B lecture 8 page 1 © copyright Bruce Blumberg 2004. All rights reserved BioSci 145B Lecture #8 5/25/2004 Bruce Blumberg –2113E McGaugh Hall -