bam ii  (ZeptoMetrix corporation)

Journal: BMC Molecular Biology

Article Title: Stringent and reproducible tetracycline-regulated transgene expression by site-specific insertion at chromosomal loci with pre-characterised induction characteristics

doi: 10.1186/1471-2199-8-30

Figure Lengend Snippet: Cre-mediated insertion by ScIn-2 . A . Schematic (not to scale) of the target locus in clone Rht14-19 or Rht14-10 before (a) and after (b) Cre-mediated insertion of pIN-2, and after flp-mediate deletion (c). DNA is represented as in Fig. 1 with recognition sites for Hind III ( H ) and Bgl II ( B ) and PCR primers indicated. B . Ethidium bromide stained agarose gel of electrophoresed PCR products generated with the primers O1 and O3 and cell pellets of 9 MPA/X r , GFP-negative clones selected after transfection of Rht14-10 (Rht14-10IN) or of Rht14-19 (Rht14-19IN) with pIN-2 and pMC-Cre. M = marker DNA. Negative (-) and positive (+) controls used, respectively, no DNA and pTIGHTgpt DNA (expected product: 555 bp). C . Southern blots of genomic DNA isolated from Rht14-10 and derivatives and digested with Bgl II (left), or from Rht14-19 and derivatives and digested with Hind III (right). Derivatives before (10IN5 and 19IN3, 4 and 5) and after (10flp9.2 and 3 and 19flp9.2 and 12.3) Flp-mediated deletion are analysed. The probe was a fragment of the d2EGFP gene (methods).

Article Snippet: The gpt ORF was removed from pBSgpt [ ] as a Bam HI/ Bgl II fragment and cloned into the Bgl II site of pGL3-Basic (Clontech) to make pGL3gptluc.

Techniques: Staining, Agarose Gel Electrophoresis, Generated, Clone Assay, Transfection, Marker, Isolation