beh.109: laboratory fundamentals in biological engineering. module 3 eukaryotic cells as phenotypic...
TRANSCRIPT
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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
DAY 3
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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
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Sunlight
Pollution
Oxidation
FoodCigarette Smoke
DNA is constantly being damaged by endogenous
and exogenous agents
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DNA Repair Strategies• Direct Reversal
Photolyase, Methyltransferase, Oxidative demethylase
• Excision RepairBase excision, nucleotide excision, transcription
coupled excision repair, mismatch repair
• Lesion AvoidanceTranslesion synthesis, DNA recombination
• Double strand break repairHomologous recombination, Non-homologous end
joining
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DNA Repair Strategies• Direct Reversal
Photolyase, Methyltransferase, Oxidative demethylase
• Excision RepairBase excision, nucleotide excision, transcription
coupled excision repair, mismatch repair
• Lesion AvoidanceTranslesion synthesis, DNA recombination
• Double strand break repairHomologous recombination, Non-homologous end
joining
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Excision Repair
Recognition
Excision
Resynthesis
Ligation
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DNA Repair Strategies• Direct Reversal
Photolyase, Methyltransferase, Oxidative demethylase
• Excision RepairBase excision, nucleotide excision, transcription
coupled excision repair, mismatch repair
• Lesion AvoidanceTranslesion synthesis, DNA recombination
• Double strand break repairHomologous recombination, Non-homologous end
joining
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REPLICATION FIDELITY
• How many times does the replicative polymerase have to choose the correct nucleotide during one cell division???
• Is one mistake in a million choices acceptable?
• How is fidelity achieved?
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Humans who have
inefficient Mismatch Repair are
highly prone to colorectal
and other cancers!!
GC
GT
G
Mismatchrecognition
MSH2 MSH6
MLH1
PMS2
Endonuclease?
GT
EXO1
PCNA, Pol , RPALigase?
GT
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DNA Repair Strategies• Direct Reversal
Photolyase, Methyltransferase, Oxidative demethylase
• Excision RepairBase excision, nucleotide excision, transcription
coupled excision repair, mismatch repair
• Lesion AvoidanceTranslesion synthesis, DNA recombination
• Double strand break repairHomologous recombination, Non-homologous end
joining
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Base Excision Repair
AAG/MPG
3MeA DNA glycosylase
initiates repair of replication
blocking lesions
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Human AAG enzyme
bound to substrate
(aka MPG)
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The absence of the AAG enzyme renders mouse
cells very sensitive to the toxic effects of
alkylating agents that damage DNA
You will try to knock down
human AAG with siRNA!!
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DNA Repair Strategies• Direct Reversal
Photolyase, Methyltransferase, Oxidative demethylase
• Excision RepairBase excision, nucleotide excision, transcription
coupled excision repair, mismatch repair
• Lesion AvoidanceTranslesion synthesis, DNA recombination
• Double strand break repairHomologous recombination, Non-homologous end
joining
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The eukaryotic cell cycle
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Three pronged
attack
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P53 has 18 sites for modification by
phosphorylation, acetylation, sumolation
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How do deficiencies in
p53
ATM
ATR
Affect human health?
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p53
Li-Fraumeni Syndrome
Germ line inheritance of mutated p53 genes
Cancer Prone
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THE FIRST SIGNS of ataxia telangiectasia (A-T) usually
appear in the second year of life as a lack of balance and slurred
speech. It is a progressive, degenerative disease
characterized by cerebellar degeneration, immunodeficiency,
radiosensitivity (sensitivity to radiant energy,
such as x-ray) and a predisposition to cancer.
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Ataxia Telangiectasia – Cancer Prone
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