BEH.109: Laboratory Fundamentals in Biological Engineering. MODULE 3 Eukaryotic Cells as Phenotypic Indicators: The use of RNAi to modulate gene expression DAY 3Slide 2Slide 3Slide 4Slide 5Slide 6Slide 7Slide 8REPLICATION FIDELITYSlide 10Slide 11Humans who have inefficient Mismatch Repair are highly prone to colorectal and other cancers!!Slide 13Slide 14Slide 15Slide 16Slide 17Slide 18Slide 19Slide 20Slide 21Slide 22Slide 23Slide 24Slide 25Slide 26Slide 27p53Slide 29Slide 30Slide 31Slide 32BEH.109: Laboratory Fundamentals in Biological Engineering. MODULE 3Eukaryotic Cells as Phenotypic Indicators:The use of RNAi to modulate gene expressionDAY 3Monday 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 weeksWe will eliminate the expression of six different genes using RNAi technology, human cells, fluorescent proteins and DNA microarraysSunlightPollutionOxidationFoodCigarette SmokeDNA is constantly being damaged by endogenous and exogenous agentsDNA Repair Strategies• Direct ReversalPhotolyase, 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 joiningDNA Repair Strategies• Direct ReversalPhotolyase, 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 joiningExcision RepairRecognitionExcisionResynthesisLigationDNA Repair Strategies• Direct ReversalPhotolyase, 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 joiningREPLICATION 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?Humans who have inefficient Mismatch Repair are highly prone to colorectal and other cancers!!GCGTGMismatchrecognitionMSH2MSH6MLH1PMS2Endonuclease?GTEXO1PCNA, Pol , RPALigase?GTDNA Repair Strategies• Direct ReversalPhotolyase, 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 joiningBase Excision RepairAAG/MPG 3MeA DNA glycosylase initiates repair of replication blocking lesionsHuman AAG enzyme bound to substrate (aka MPG)The absence of the AAG enzyme renders mouse cells very sensitive to the toxic effects of alkylating agents that damage DNAYou will try to knock down human AAG with siRNA!!DNA Repair Strategies• Direct ReversalPhotolyase, 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 joiningThe eukaryotic cell cycleThree pronged attackP53 has 18 sites for modification by phosphorylation, acetylation, sumolationHow do deficiencies inp53ATMATRAffect human health?p53Li-Fraumeni SyndromeGerm line inheritance of mutated p53 genesCancer ProneTHE 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.Ataxia Telangiectasia – Cancer