Module 2: Expression EngineeringModule 2: Expression Engineering20.109Lecture 3October 18th, 2007Cite as: Natalie Kuldell. Course materials for 20.109 Laboratory Fundamentals in Biological Engineering, Fall 2007. MITOpenCourseWare (http://ocw.mit.edu), MassachusettsInstitute of Technology. Downloaded on [DD Month YYYY].STEM Educationbest way to achieve Adequate Yearly Progress?Content removed due to copyright restrictions. Excerpt from Medina, Jennifer. “Making Cash a Prize for High Scores on Advanced Placement Tests.”The New York Times, October 15, 2007. [http://www.nytimes.com/2007/10/15/nyregion/15rewards.html, accessed May 8, 2008.]Excerpt from Kula, Adam and Alexandra Frean. “Make science easier, examiners are told.” The Times, August 29, 2007. [http://www.timesonline.co.uk/tol/news/uk/education/article2344054.ece, accessed May 8, 2008.]Cite as: Natalie Kuldell. Course materials for 20.109 Laboratory Fundamentals in Biological Engineering, Fall 2007. MITOpenCourseWare (http://ocw.mit.edu), MassachusettsInstitute of Technology. Downloaded on [DD Month YYYY]. Excerpt from Cavanaugh, Sean. "Federal Rule Yields Hope for Science." Education Week, published online October 5, 2007.Expression Engineering ExperimentDay 1Day 2Day 3Image of glowing luciferasein a microcentrifuge tube, removed due to copyright restrictions.RTFigure by MIT OpenCourseWare.Day 6Day 5 Day 4Image credits: Day 1 - Courtesy of Arkitek Studios. Used with permission. Day 2 - Courtesy of The Exploratorium. © The Exploratorium, http://www.exploratorium.edu. Day 4 – Figure by MIT OpenCourseWare. Day 5 – Dr. Natalie Kuldell. Day 6 – Courtesy of NIH.Cite as: Natalie Kuldell. Course materials for 20.109 Laboratory Fundamentals in Biological Engineering, Fall 2007. MITOpenCourseWare (http://ocw.mit.edu), MassachusettsInstitute of Technology. Downloaded on [DD Month YYYY].Expression Engineering ExperimentLecture 1• intro to cell culture• intro to gene exp’n/RNAiLecture 2• transfection• luciferaseLecture 3• off-target/nonspecific RNAiLecture 4• Writing lecture(Neal Lerner)Lecture 5• measuring gene express’nLecture 6• microarray analysis (Rebecca Fry)Lecture 7• high throughput technologies or RNAi applications (no lab)Lecture 8• review of your dataCite as: Natalie Kuldell. Course materials for 20.109 Laboratory Fundamentals in Biological Engineering, Fall 2007. MITOpenCourseWare (http://ocw.mit.edu), MassachusettsInstitute of Technology. Downloaded on [DD Month YYYY].RNAiOperation or Battleship?Images removed due to copyright restrictions. Photos of the games “Operation” and “Battleship.”Cite as: Natalie Kuldell. Course materials for 20.109 Laboratory Fundamentals in Biological Engineering, Fall 2007. MITOpenCourseWare (http://ocw.mit.edu), MassachusettsInstitute of Technology. Downloaded on [DD Month YYYY].Where were you in 1998?Content removed due to copyright restrictions. News-related images:• “France wins the World Cup”• “Clinton under Investigation” [re: Monika Lewinski]• President Clinton speaking at MIT commencement• Movie poster for “Titanic”Cite as: Natalie Kuldell. Course materials for 20.109 Laboratory Fundamentals in Biological Engineering, Fall 2007. MITOpenCourseWare (http://ocw.mit.edu), MassachusettsInstitute of Technology. Downloaded on [DD Month YYYY].Image removed due to copyright restrictions.Excerpt from Table 1: Effects of sense, antisense and mixed RNAs on progeny of injected animals. In Fire, A., et al. “Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.” Nature 391 (19 February 1998). doi:10.1038/35888.Source: NIH.Cite as: Natalie Kuldell. Course materials for 20.109 Laboratory Fundamentals in Biological Engineering, Fall 2007. MITOpenCourseWare (http://ocw.mit.edu), MassachusettsInstitute of Technology. Downloaded on [DD Month YYYY].small-RNA revolution# of publications:~15 in 1998 ~1000 in 2000~ 4500 in 2007 (so far)Image removed due to copyright restrictions.Cover of Science 298 no. 5602 (20 December 2002).“New roles for RNAs.”Cite as: Natalie Kuldell. Course materials for 20.109 Laboratory Fundamentals in Biological Engineering, Fall 2007. MITOpenCourseWare (http://ocw.mit.edu), MassachusettsInstitute of Technology. Downloaded on [DD Month YYYY].RNA interference vocabularyTransfection DicerRISCsiRNACite as: Natalie Kuldell. Course materials for 20.109 Laboratory Fundamentals in Biological Engineering, Fall 2007. MITOpenCourseWare (http://ocw.mit.edu), MassachusettsInstitute of Technology. Downloaded on [DD Month YYYY]. Courtesy of Arkitek Studios. Used with permission.Source: RNAi Interface animation for nature.com (http://www.nature.com/focus/rnai/animations/index.html)Nomenclature for siRNAs5’-PO45’-PO43’-OH3’-OH+1+19TTTTRNADNA template“coding strand” “template strand”ATG TAATAC ATTAUG UAA“sense”Cite as: Natalie Kuldell. Course materials for 20.109 Laboratory Fundamentals in Biological Engineering, Fall 2007. MITOpenCourseWare (http://ocw.mit.edu), MassachusettsInstitute of Technology. Downloaded on [DD Month YYYY].Nomenclature for siRNAsTTRNADNAATG TAATAC ATTTT“sense”TTRISC“antisense”ATG TAATAC ATTAUG UAACite as: Natalie Kuldell. Course materials for 20.109 Laboratory Fundamentals in Biological Engineering, Fall 2007. MITOpenCourseWare (http://ocw.mit.edu), MassachusettsInstitute of Technology. Downloaded on [DD Month YYYY].Nomenclature for siRNAsTTRNADNATTRISCATG TAATAC ATTAUG UAATTTTTTCite as: Natalie Kuldell. Course materials for 20.109 Laboratory Fundamentals in Biological Engineering, Fall 2007. MITOpenCourseWare (http://ocw.mit.edu), MassachusettsInstitute of Technology. Downloaded on [DD Month YYYY].See “Post-transcriptional gene silencing by dsRNA.”Nature Reviews Genetics 2001; 2:110-119.Delivery vs expression of dsRNAMethoddsRNAdsRNAdsRNAStem-loopexpressionDual promoter5'3'5'5'3'3'VirusPlantsTrypanosomesC. elegansDrosophilaTrypanosomesPlantsC. elegansDrosophilaTrypanosomesOrganismMethods of delivering double-stranded RNAFigure by MIT OpenCourseWare.Cite as: Natalie Kuldell. Course materials for 20.109 Laboratory Fundamentals in Biological Engineering, Fall 2007. MITOpenCourseWare (http://ocw.mit.edu), MassachusettsInstitute of Technology. Downloaded on [DD Month YYYY].fig from Svoboda review in Curr Opin Mol Ther. 2007 9(3):248RNAi: siRNA vs miRNARISC biochemistry unites RNAiFigure removed due to copyright restrictions.