Pdx1 Cre-ER (DM#255)
(Plasmid #15022)

• Depositing Lab: Douglas Melton
• Publication: Gu et al Development. 2002 May . 129(10):2447-57.

Backbone

• Vector backbone: pBluescript II KS
• Backbone size w/o insert (bp): 3000
• Vector type: Mammalian Expression, Cre/Lox ; mouse transgenic

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): Stbl3
• Growth instructions: Use Stbl3 to prevent recombination
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: Pdx1 promoter
• Alt name: pGD35
• Species: M. musculus (mouse)
• Insert Size (bp): 10000
• Entrez Gene: Pdx1 (a.k.a. IDX-1, IPF-1, Ipf1, Mody4, STF-1, pdx-1)
• Tag / Fusion Protein:
  • Cre-ER(TM) (C terminal on insert)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ sequencing primer: T7
• 3′ sequencing primer: T3

Resource Information

• A portion of this plasmid was derived from a plasmid made by: Cre-ER(TM) was a gift from Andy McMahon.

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
• Industry Terms:
  • Not Available to Industry

Depositor 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.