Lecture 15 - Genetic Engineering

cattlejoyousBiotechnology

Dec 10, 2012 (4 years and 10 months ago)

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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  
Cell  Membrane  Nucleus  
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  
nobelprize.org  
Further  inbreeding  leads  to  
homozygous  (double  KO)  
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  
loxP  loxP  Stop  Promoter  
Gene  OI  
 +  Cre  
loxP  Promoter  
Gene  OI  
Stop  
loxP  
 +  Cre  
Promoter  Gene  OI  
loxP  
loxP  
Promoter  Gene  OI  
loxP  
loxP  
Cre-­‐expressing  cell  +  floxed  viral  
construct  
Promoter  Gene  OI  
loxP  
loxP  
Cre  expressing  Non-­‐Cre  expressing  
Infect  
Cre  expressing  Non-­‐Cre  expressing  
Inac-ve  gene  Ac-ve  gene  
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)