BEH.109: Laboratory Fundamentals in Biological Engineering. MODULE 3 Eukaryotic Cells as Phenotypic Indicators: The use of RNAi to modulate gene expression Instructor: Leona D. Samson Teaching Assistants: Jenn Cheng and Lisa Joslin With additional invaluable help from Lisa Smeester and Rebecca Fry
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BEH.109: Laboratory Fundamentals in Biological Engineering.
MODULE 3
Eukaryotic Cells as Phenotypic Indicators:
The use of RNAi to modulate gene expression
Instructor: Leona D. SamsonTeaching Assistants: Jenn Cheng and Lisa
Joslin
With additional invaluable help from Lisa Smeester and Rebecca Fry
Monday March 31 DAY 1
Module 3 Overview & mini-lecture on RNAi Safety Orientation Sterile Technique Transfection of EGFP & p53 siRNA into EGFP expressing HeLa cells
Tues April 1 DAY 1
Module 3 Overview & mini-lecture on RNAi Safety Orientation Sterile Technique Transfection of EGFP & p53 siRNA into EGFP expressing HeLa cells
Wed April 2 DAY 2
Comprehensive lecture on RNAi with some examples Harvest transfected cells Microscope analysis & FACS analysis Analyze data
Thurs April 3 DAY 2
Comprehensive lecture on RNAi with some examples Harvest transfected cells Microscope analysis & FACS analysis Analyze data
Monday April 7 DAY 3
Introduction to the ATM, ATR, EXO1 and AAG genes Ambion and Blast session to design new siRNAs for four genes. siRNA is ordered for next experiment
Tues April 8 DAY 3
Introduction to the ATM, ATR, EXO1 and AAG genes Ambion and Blast session to design siRNAs for four genes. siRNA is ordered for next experiment
Wed April 9 DAY 4
Introduction to DNA microarrays and overview of what will happen on days 5 & 6 Transfect four new si.RNAs; cellular RNA will be isolated over the w/e Informal Presentation of FACS data by students
Thurs April 10 DAY 4
Introduction to DNA microarrays and overview of what will happen on days 5 & 6 Transfect four new si.RNAs; cellular RNA will be isolated over the w/e Informal Presentation of FACS data by students
Monday April 14 DAY 5
Label isolated RNA and hybridize to microarray slides
Tues April 15 DAY 5
Label isolated RNA and hybridize to microarray slides
Wed April 16 DAY 6
Scan microarray slides and analyze results
Thurs April 17 DAY 6
Scan microarray slides and analyze results
Patriots Day
MIT Holiday
Wed April 23 DAY 7
MODULE 3 Student Presentations
Thurs April 24 DAY 7
MODULE 3 Student Presentations
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 3Day 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”
Arabidopsis thaliana
Planaria
Trypanosomes Hydra
Protozoa can use RNAi for gene
silencing
RNAi can operate in
insects too!
Sulston and Horvitz (1977). Develop. Biol. 56, 110-156.
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
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!
19 nt duplex
2 nt 3’ overhangs
C. Elegans movie
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