Application

EMD Millipore has developed a cell-permeant TAT-CRE Recombinase fusion protein which can be directly delivered to mammalian cells and results in high recombination efficiencies (75 – 100% in mouse and ~ 60% in human cells). TAT-CRE readily translocates to mammalian cells and can catalyze highly efficient recombination. The dose and timing of Cre exposure can be precisely controlled thus allowing for the careful titration of Cre activity.

General description

Cre Recombinase is an enzyme from bacteriophage P1 that catalyzes the site-specific recombination between two DNA recognition sites termed loxP sites. The LoxP recognition site consists of two 13 bp inverted repeats flanking a 8 bp spacer region. Because the Cre gene and loxP sites are not native to the genomes of most species, LoxP sites can be engineered and introduced into target cells and thus used as a means to precisely control the expression of genes in vitro (i.e. cultured cells) and in vivo (i.e. animal models).

If LoxP sites are located on different chromosomes, Cre recombinase will mediate a chromosomal translocation.
If LoxP sites are oriented in the opposite direction, Cre recombinase will mediate an inversion of the floxed segment.
If LoxP sites are oriented in the same direction, Cre recombinase will mediate a deletion of the floxed segment.

In this way, placement of the LoxP sites allows genes to be activated, repressed or exchanged for other genes.

EMD Millipore's TAT-CRE Recombinase is a recombinant cell-permeant fusion protein consisting of a basic protein translocation peptide derived from HIV-TAT (TAT), a nuclear localization sequence (NLS), the Cre protein and an N-terminal histidine tag (H6) for efficient purification of the protein from E. coli.

EMD Millipore's TAT-CRE Recombinase has been shown to effectively excise STEMCCA viral transgenes from both Human and Mouse IPS cells.

Physical form

Recombinant protein is supplied in buffer containing 50% glycerol (v/v) 500 mM NaCl and 20 mM HEPES at pH 7.4

Quality

Each lot of TAT-CRE Recombinase protein is rigorously quality control tested for the following parameters:
Purity: single band around 41 kDa with greater than 70% protein purity on an SDS-PAGE gel
Functional activity: mediates recombination of LoxP-modified alleles in a HEK293T- Cre reporter cell line
Endotoxin levels: less than 1 EU/ug protein
Mycoplasma negative

Storage and Stability

Stable for 3 months from date of receipt when stored at -20°C or -80°C. Upon first thaw, centrifuge the vial and gently mix the solution. Aliquot into smaller working volumes and freeze at -20°C or -80°C. Before use, dilute TAT-CRE to the appropriate concentration with culture medium and filter through a 0.2um low protein binding syringe filter (Millipore Cat. No. SLGV 033RS or SLGV013 SL).

Unit Definition

A standard of 100 Units is defined as the amount of TAT-CRE (ug) in 1.0 mL of tissue culture medium that is required to induce 50% GFP expression in a HEK293T loxp reporter cell line assay.