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URL: http://www.addgene.org/53126

Proper Citation: RRID:Addgene_53126

Insert Name: Gfa2

Organism: Homo sapiens

Bacterial Resistance: Ampicillin

Defining Citation: PMID:8120611

Vector Backbone Description: Vector Backbone:pUC18; Vector Types:Mammalian Expression, Mouse Targeting; Bacterial Resistance:Ampicillin

Comments: The plasmid is a pUC18 vector containing the human GFAP sequences from -2163 to +47 (the human gfa2 segment), with the initiating ATG mutated to TTG, placed in front of the nuclear targeted E. coli lacZ gene, which in turn is followed by a fragment of the mouse protamine-1 gene. The latter supplies an intron, stabilizing 3' UTR, and a polyadenylation signal. If cloning a cDNA, it is advisable to retain the mP-1 segment. The lacZ gene can be excised by digestion with BamHI, and replaced with your gene of interest. If cloning a genomic sequence that includes an intron and polyadenylation site (this may compromise astrocyte specificity--see Su et al. 2004), the mP-1 region can also be excised, or alternatively, a number of sites flank the gfa2 segment allowing its isolation. Restriction sites that may be useful are as follows: EcoRI: flanks the gfa2, lacZ, and mP-1 segments Bgl II: 5' flank of gfa2 & 3' flank of mP-1 Bam HI: flanks the lacZ gene Sal I: cuts uniquely between the gfa2 promoter and the LacZ gene Sph I and Hind III: cut uniquely just 3' of the mP-1 segment Although the GFAP promoter appears to be the best choice for targeting transgene expression to astrocytes in mice, please be aware that neuronal expression has also occasionally been observed (Su et al. 2004).

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