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BEH.109: Laboratory Fundamentals in Biological Engineering. MODULE 3Eukaryotic Cells as Phenotypic Indicators:The use of RNAi to modulate gene expressionInstructor: Leona D. SamsonTeaching Assistants: Jenn Cheng and Lisa JoslinWith additional invaluable help from Lisa Smeester and Rebecca Fry
Snapshot of the next four weeks We will eliminate the expression of six different genes using RNAi technology, human cells, fluorescent proteins and DNA microarrays
You WILL be required to write a lab report, but Class still happens on April 24rd. Please follow the "BEH.109 Guidelines for Module Reports"handout that was given to you previously. The report will be DUE ON MONDAY, APRIL 29th BY 5 PM.
RNA Interference - RNAi BE109 Module 3 Day 2 lecture
RNA interference first discovered in Petunias! Called PTGS, for “Post Transcriptional Gene Silencing”
Color changes can be induced by RNAi, or PTGS.. Post transcipt-ional gene silencing
Small (21-23 nts) RNA duplexes, with the same sequence as in the silenced gene, were identified as being responsible for knocking down expression
So what other organisms can do this thing called PTGS? “Post Transcriptional Gene Silencing”
Protozoa can use RNAi for gene silencing Planaria Trypanosomes Hydra
RNAi is used by C. elegans to control the timing of development of various tissues Such gene silencing is a natural phenomenon in this organism
19 nt duplex 2 nt 3’ overhangs This dsRNA species found in plants, C. elegans and Drosophila melanogaster undergoing gene silencing….but how to prove it is responsible? Purified them and showed in vitro silencing in Drosophila extracts; used sythetic sdRNA oligo to achieve same thing!
C. Elegans grow on agar dishes with E. coli bacteria as a source of food. If they eat E. coli expressing dsRNA molecules…this creates specific knock-down of gene expression!
In C. elegans the siRNA effects can be amplified making the silencing quite stable This does not appear to happen in mammalian cells (RISC = RNAi Induced Silencing Complex; RdRP = RNA dependent RNA polymerase)
http://www.nature.com/cgi-taf/DynaPage.taf?file=/nature/journal/v421/n6920/full/421220a_fs.html&content_filetype=pdfhttp://www.nature.com/cgi-taf/DynaPage.taf?file=/nature/journal/v421/n6920/full/421220a_fs.html&content_filetype=pdf
19,757 genes 16,757 have been inactivated by RNAi 10% display spontaneous phenotype; this 10% is enriched for conserved genes
19,757 genes 16,757 knock down mutants were screened for body fat content 305 knock downs had increased body fat, 112 genes had decreased..new targets for obesity?
How does RNAi work in mammalian cells? RNAi works postranscriptionally…….. in key two steps!
Model for RNAi siRNA
step one: 34 27 21 20 16 short-interfering RNA processing the dsRNA into 21-23 nt fragments Tuschl, 2001
Dicer contains two RNAse III domains long dsRNA siRNAs
19 nt duplex 2 nt 3’ overhangs siRNAs have a defined structure
step two: the antisense strand of the siRNA guides cleavage Tuschl, 2002
RNAi silencing complex • may be associated with translating ribosomes • active RNAse enzyme not yet identified • may participate in endogenous pathways that silence genes via translational repression
P P interferon production PKR eiF2a P apoptosis Blockage of protein synthesis Mammals exhibit potent responses to dsRNA dsRNA cell death
smaller RNAs can escape the PKR pathway recall that siRNAs are intermediate effectors In the RNAi pathway siRNAs are not recognized by the PKR!
lentiviral construct for siRNAs Rubinson et al Nature Genetics, 2003
biological research defining gene function (gene knockout) C. elegans genome RNAi projects defining biochemical pathways microarray screening of RNAi knockouts therapeutic treatment cancer viral infection parasitic infection Practical Aspects of RNAi
What is the killer virus that might sweep the globe? Will RNAi eventually become an effective and specific antiviral therapy?
