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• 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
• 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
• 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)
• 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
• 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
• 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
• 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
• 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
• 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
• 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
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
• 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
• 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