pWHERE

pWHERE plasmid was designed for studies of temporal expression and tissue distribution of your promoter of interest, cloned within an insulated LacZ cassette, in transgenic mice and rats. A multiple cloning site (MCS) has been added upstream of the LacZ gene for convenient cloning of your promoter of interest.

pWHERE can be purchased alone or as part of a kit containing the pWHERE control plasmid with the rat EF-1α promoter.

Specifications

- LacZ transcription unit is flanked on each side by murine H19 insulators
- CpG-free LacZ Reporter Gene
- Convenient insertion of your promoter of interest
- Easy Excision of the E. coli region
- Efficient in vivo arrest of the transgene transcription

Contents

The pWHERE plasmid is provided as 20 µg of lyophilized DNA. Plasmid is shipped at room temperature. Store at -20°C.

The pWHERE plasmid is also available in a kit that contains:
- 20 µg pWHERE plasmid
- 10 µg pWHERE-rEF1 containing the rat EF-1α promoter
 

Description

Insulation of the LacZ Transcription Unit
The LacZ transcription unit is flanked on each side by two murine H19 insulators. The insulators are expected to protect the integrated transcriptional LacZ unit from negative as well as positive influences from neighboring sequences. Insulator elements can be functionally identified by their ability to shield promoters from regulators in a position-dependent manner or by their ability to protect adjacent transgenes from position effects. The fragment of the differential methylated region (DMD) located between the mouse Igf2 and H19 acts as a powerful insulator [1].
The enhancer blocking activity of the DMD fragment is dependent upon four responsive elements to the vertebrate enhancer-blocking protein CTCF [2]. Two mouse DMD fragments have been introduced in the pWHERE plasmid to insulate the expression cassette, containing your promoter of interest, from the 5‘ and 3‘ adjacent regions at the integrated site in transgenic mice.

CpG-free Reporter Gene
The pWHERE plasmid contains the lacZ∆CpGnls gene, a chemically synthesized new allele, free of CpGs to eliminate interference of CpG methylation on expression.

Convenient Insertion of Your Promoter of Interest
A multiple cloning site with many restriction sites is located upstream of the lacZ∆CpGnls gene for convenient cloning of your own promoter or a promoter chosen from InvivoGen's  pDRIVE list.
MCS 5‘- Sda I, Avr II, Bam HI, Xho I, Sma I, Nco I -3‘

Easy Excision of the E. coli Region
The E. coli region is flanked on either side by the efficient 8 bp-recognizing restriction enzyme Pac I.

Efficient in vivo Arrest of the Transgene Transcription
This feature is achieved by the UTR and polyadenylation sequence of the human elongation factor 1-alpha gene.

1. Kaffer CR. et al. 2000. A transcriptional insulator at the imprinted H19/Igf2 locus. Genes Dev. 14:1908-19
2. Bell AC. and Felsenfeld G. 2000. Methylation of a CTCF-dependent boundary controls imprinted expression of the Igf2 gene. Nature. 405:482-485