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URL: http://www.addgene.org/15022

Proper Citation: RRID:Addgene_15022

Insert Name: Pdx1 promoter

Organism: Mus musculus

Bacterial Resistance: Ampicillin

Defining Citation: PMID:11973276

Vector Backbone Description: Backbone Size:3000; Vector Backbone:pBluescript II KS; Vector Types:Mammalian Expression, Cre/Lox, mouse transgenic; Bacterial Resistance:Ampicillin

Comments: The CRE-ER(TM) protein requires tamoxifen (TM) to catalyze LoxP site-mediated excision. For Pdx-1-Cre-ER(TM), the coding region of the Cre-ER(TM) cDNA was directly fused to the starting ATG of the PDX1 protein by PCR. Three primers (p5, 5'-ttgaaacaagtgcaggtgttcg-3'; p75, 5'-gttgcatcgaccggtaatgcaggcaaattttggtgtacggtcagtaaattggacatggtggcagccggcact-3'; and p71, 5'-gttgcatcgaccggtaatgca-3') were used. First, p5 and p75 were used to amplify a 400 base pair fragment from the Pdx1 genomic DNA. Then this fragment was used together with p5 and p71 and the Cre-ER(TM) plasmid to obtain a fragment that has the 5' end coding region directly fused to the Pdx1 promoter. This fragment was digested with AgeI and ligated to the XhoI (blunt-ended)-AgeI double-digested pBSCre-ER(TM) that contains the full-length Cre-ER-coding region to give pGD19. Then a 2.2 kb insert was released from pGD19 by SmaI-SpeI digestion and ligated to SmaI-NotI digested pKSpdx-1SalI, and a SpeI-NotI digested PCR fragment that contains the SV40 polyadenylation signal to give pGD35. The 8 kb insert was released by SalI-NotI digestion and was used for pronucleus injection. See Author's Map and article for more information. SalI/SmaI digest should give 6.7, 1.5, 4 kb bands.

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