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• Outline - 1 point• Actual paper – 5 pages single spaced 1” margins (references not
included). • 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. After each one, use a sentence or two to state what you will do to answer these questions
– Finish with a paragraph stating what will be the significance of the research assuming that you successfully execute the proposed experiments.
– 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 among the most important parts of any grant application. You have to convince the reviewer here that your work is important and worth funding.
• 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
• 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)• http://blumberg-lab.bio.uci.edu/bioD145-w2015/example_grant.pdf
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.
• 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
• 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
• 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)
• 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 (Boutros, Luo papers this week)– Nuclease based methods - introduce double-stranded breaks
• 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
• 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
• viruses and transposable elements can deliver DNA to random locations– can disrupt gene function – put inserted gene under the
Insertional mutagenesis by the Tol2 transposon-mediated enhancer trap approach generated mutations in two developmental genes: tcf7 and synembryn-like. Nagayoshi S, Hayashi E, Abe G, Osato N, Asakawa K, Urasaki A, Horikawa K, Ikeo K, Takeda H, Kawakami K. Development 2008 Jan;135(1):159-69.