Lecture 15 - Genetic Engineering

Lecture c+10 Gene-c engineering for neuroscience Making the brain fabulous Lichtman Lab Proteins to record neural ac-vity GCaMP Proteins to control neural ac-vity Fenno LE, et al., Annual Review of Neuroscience. 2011, 34:389. Fenno LE, et al., Annual Review of Neuroscience. 2011, 34:389. Protein synthesis – central dogma Wikipedia.org Expressing ar-ficial proteins Microinjec-on Viral Vectors Lipofec-on Electropora-on Nucleus Cell Membrane Crea-ng animals with ar-ficial genes Central Dogma of Biology Promoter + Transcrip-on factors determine when/where genes are expressed Lucchessi lab, Emory Homologous recombina-on Wikipedia.org Introducing a transgene Wikipedia.org Further inbreeding leads to homozygous (double KO) nobelprize.org wikipedia.org Tissue specific expression •  Find genes that are expressed in specific brain areas. •  Design DNA constructs that will be integrated under the same promoter. The internet: mouse.brain-­‐map.org Thy1 vs RGS9 IER5 vs. TCF7L2 Transgenic animals GeneraOng transgenic animals can take many months or longer… CRE – recombinase Nagy, A. et al. Genesis, 26, 99 (2000) Cre-­‐LoxP system LoxP sequence bound by Cre Jackson Laboratory Cre-­‐dependent expression Stop loxP Gene OI loxP + Cre Promoter loxP Gene OI Stop loxP Promoter Gene OI Promoter loxP loxP + Cre loxP Promoter Gene OI loxP Cre-­‐expressing cell + floxed viral construct Cre expressing Non-­‐Cre expressing Cre expressing Non-­‐Cre expressing Ac-ve gene Inac-ve gene Infect Promoter Gene OI loxP loxP Eg. Thy-­‐1 Cre jaxmice.jax.org/strain/006143.html The Brainbow RGB colorspace Three fluorophores can encode many colors CFP GFP RFP Brainbow 1.0 & 1.1 Livet et al. Nature 450, 56 (2007). •  Today: –  Gene-c engineering to target protein expression by cell type (and/or brain region) •  Next -me: –  3d imaging in the brain (mulit-­‐photon microscopy) –  Neural circuit dynamics (synap-c plas-city)