Identification of cell-type-specific promoters within the brain using lentiviral vectors.
The development of cell-type-specific mini-promoters for genetic studies is complicated by a number of issues. Here, we describe a general method for the relatively rapid screening of specific promoter activity in cell culture, in acute brain slice preparations and in vivo. Specifically, we examine the activity of an approximately 3 kb promoter region from the neuroactive peptide cholecystokinin (CCK) compared to the commonly used cytomegalovirus promoter. We find a high degree of cell-type selectivity in vivo using lentiviral approaches in rats and traditional transgenic approaches in mice. Appropriate colocalization of Cre-recombinase and CCK gene expression is found within the hippocampus, when the CCK promoter is driving either the expression of Cre-recombinase or green fluorescent protein. We also demonstrate fluorescent identification of CCK-positive interneurons that allows for cell-type-specific electrophysiologic studies in rats and mice. In conclusion, these studies identify a functional mini-promoter for the CCK gene and outline a novel and sensitive general method to test activity of selective promoters in vitro and in vivo. This approach may allow for the more rapid identification of specific promoters for use with transgenic animals, in genetically modified viruses, and in the design of targeted, therapeutic gene-delivery systems.
Chhatwal
J P
JP
Department of Psychiatry and Behavioral Sciences, Center for Behavioral Neuroscience, Yerkes National Primate Research Center, Emory University, Atlanta, GA 30329, USA.
Hammack
S E
SE
Jasnow
A M
AM
Rainnie
D G
DG
Ressler
K J
KJ
D000818:Animals; D001921:Brain / Q000378:metabolism*; D002766:Cholecystokinin / Q000235:genetics; D015870:Gene Expression; D017930:Genes, Reporter; D005818:Genetic Engineering; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics* / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics; D019426:Integrases / Q000235:genetics; D016086:Lentivirus / Q000235:genetics*; D051379:Mice; D008822:Mice, Transgenic; D008856:Microscopy, Fluorescence; D011401:Promoter Regions, Genetic; D051381:Rats; D017207:Rats, Sprague-Dawley; D014161:Transduction, Genetic / Q000379:methods; D019076:Transgenes
D049452:Green Fluorescent Proteins; D002766:Cholecystokinin; C045073:Cre recombinase; D019426:Integrases