Identification of cell-type-specific promoters within the brain using lentiviral vectors

Abstract

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 B3 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 genedelivery systems.

Prelimbic cortical BDNF is required for memory of learned fear but not extinction or innate fear

“…To examine the role of cortex-specific BDNF in modulating fear, we used a newly developed cortex-specific BDNF knockout mouse model (33). Expression of Cre recombinase in these mice includes the PL and neocortex but spares the more ventral IL, along with all other paleocortical and subcortical regions, with no expression in hippocampus, thalamus, striatum, hypothalamus, or amygdala ( Fig.…”

“…Lentivirus experiments used homozygous BDNF-floxed mice (34) (Bdnf tm3Jae /J; Jackson Labs), which possess loxP sites both upstream and downstream of exon 5 of the BDNF gene. The cortex-specific Cre mouse line, previously described as "transgenic line C" (33), was created when the coding sequence for Cre recombinase was placed downstream of a 3-kb CCK promoter. The cortexspecific Cre line was crossed to floxed BDNF mice, floxed-stop[Jackson Labs, Gt (ROSA)26Sor] lacZ reporter mice (35), and floxed-stop EGFP-tdTomato mice (36).…”

Optical techniques in optogenetics

“…AAV-mediated expression may be less stable because a much smaller percentage of the virus is integrated. These vectors can be rendered cell type-specific by the choice of promoter [29], viral receptors [30], or spatial targeting strategies [31]. The volume and concentration of virus determines the spread in the tissue and opsin expression region.…”

Mammalian Models Based on RCAS-TVA Technique

“…Additionally, numerous studies have indicated that some other promoters, including the ovary specific promoter (OSP1) and the high-affinity folate receptors promoter (HAFR) (Godwin et al, 1995;Goldsmith et al, 1999), the modified rat probasin (rPB) promoters (Furuhata et al, 2003), and the neuroactive peptide cholecystokinin (CCK) promoter (Chhatwal et al, 2007), are of potential value for tissue specific expression of the tv-a gene. 1.2 Both isoforms of TVA molecule are sufficient for acceptance of the RCAS vectors As mentioned earlier, two isoforms, a transmembrane and a GPI-linked one, have been identified.…”