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 FrySlide 2Slide 3Central Dogma of Molecular BiologyTranscriptionWhat is mRNA?Slide 7Slide 8Slide 9Slide 10Slide 11Slide 12Slide 13Slide 14Slide 15Slide 16Slide 17Slide 18Slide 19Slide 20Slide 21Slide 22Slide 23Slide 24Slide 25Slide 26Slide 27Slide 28Slide 29Slide 30Slide 31Slide 32Slide 33Slide 34Slide 35Slide 36BEH.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 FryMonday 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 microarraysThe use of RNAi to modulate gene expressionWhy do we want to be able to modulate gene expression?Central Dogma of Molecular BiologyTranscriptionWhat is mRNA?GCU = Ala“Genes” were first described by their mutant phenotype…e.g., Mendel described inherited properties like wrinkled versus smooth peas…later Bateson coined the word “gene” to account for these phenotypic traits. Genes were said be inherited in a Mendelian fashion.1940’sBeadle and Tatum’s classic experiment with moulds established the “one gene one enzyme” hyothesisIn the 1940’s Beadle and Tatum mutated genes to analyze biochemical pathwaysMutagens?X-rays, Nitrogen MustardsNeurospora mould1940’sBeadle and Tatum’s classic experiment with moulds established the “one gene one enzyme” hyothesisEliminating the expression of a gene is one of the most powerful tools in biologyWe can now engineer the deletion of specific genes to probe their biological funtionForward Genetics:Phenotype GenotypeReverse GeneticsGenotype PhenotypeThe most common method for Reverse Geneticshas beenTargeted Gene DeletionNumber of Genes in Different OrganismsYeast ~ 6200 genes E. coli ~ 4200 genes Human ~ 30,000 genes Mouse ~ 30,000 genesPhage T4 ~ 200 genes Influenza ~ 12 genes1997 saw the sequencing of the first eukaryotic genome….S. cerevisiaeThe field is changed for ever...kanR5' 3'3’ 5'Target Gene HomologyUnique TagskanRRound 1 PCR5' 3'3 ’ 5'Round 2 PCRAUG TAA AUG TAA X XIntegration by Homologous RecombinationkanRGene Deletion Strategy- Gene Specific- G418 selection- 2 unique “bar-codes”Saccharomyces Genome Deletion ProjectWhat does knowing the sequence of all 6,200 genes enable? Making 6,200 Gene Deletion Strains!No treatment 0.01% MMS0.02% MMS0.025% MMS1 2 3 4 5 6 7 89 10 11 12 1 2 3 45 6 7 8 9 10 11 12H G F E D C B AH G F E D C B ANo treatment 0.01% MMS0.02% MMS0.025% MMS1 2 3 4 5 6 7 89 10 11 12 1 2 3 45 6 7 8 9 10 11 12H G F E D C B AH G F E D C B AGenomic Phenotyping Using MMSWTmag1rev1rad14Number of Genes in Different OrganismsYeast ~ 6200 genes E. coli ~ 4200 genes Human ~ 30,000 genes Mouse ~ 30,000 genesPhage T4 ~ 200 genes Influenza ~ 12 genesMammals are diploid!Have to knock out both genes to test the null phenotype“We will eliminate the expression of six different genes using RNAi technology, human cells, fluorescent proteins and DNA microarrays”What cells? HeLa cells What genes?Today…… EGFP..enhanced Green Fluorescent Protein gene and p53 gene as a controlNext week Four additional genesThe Origin of HeLa CellsHeLa cells in cultureHeLa cells have been cultured continuously for scientific use since they were first taken from the tumor of a woman suffering from cervical cancer in the 1950s. They have been utilized for many purposes, including the development of a polio vaccine, the pursuit of a cure for diseases such as leukemia and cancer, and the study of thecellular effects of drugs and radiation.HeLa cells from the Nikon microscope web siteMitotic HeLa cell stained with