Description:

Cre recombinase, often abbreviated to Cre, is a Type I topoisomerase from P1 bacteriophage that catalyzes site-specific recombination of DNA between loxP sites. The enzyme does not require any energy cofactors and Cre-mediated recombination quickly reaches equilibrium between substrate and reaction products. The loxP recognition element is a 34 base pair (bp) sequence composed of two 13 bp inverted repeats flanking an 8 bp spacer region which confers directionality. Recombination products are dependent on the location and relative orientation of the loxP sites. Two DNA species containing single loxP sites will be fused whilst DNA between loxP sites in the same orientation will be excised in circular form and DNA between opposing loxP sites will be inverted with respect to the rest of the DNA.

Cre recombinase is used as a tool to modify genes and chromosomes. In this approach the Cre recombinase is used to delete a segment of DNA flanked by LoxP sites (aka 'floxed') in an experimental animal. It has been used to generate animals with mutations limited to certain cell types (tissue-specific knockout) or animals with mutations that can be activated by drug administration (inducible knockout) in a number of transgenic species. The availability of transgenic lines with tissue specific or inducible Cre expression permits researchers to inactivate or activate a gene of interest simply by breeding a floxed animal to pre-existing Cre-transgenics. One example of an inducible Cre recombinase system is the Cre-ER (ER = Estrogen Receptor) system in which intraperitoneal injection of tamoxifen will cause dose-dependent excision of the floxed site (i.e. will inactivate the gene of choice).

Recombinant Cre recombinase HTNC is purified from an E. coli strain carrying an engineered plasmid encoding Cre Recombinase from bacteriophage P1 with additional N-terminal 6XHis tag, a Tat peptide (GRKKRRQRRRPPAGTSVSL) and an NLS sequence (PKKKRKV). This cell-permeant Cre recombinase (HTNC) is the most effective protein in transduction (in vivo) and subsequent recombination compared to other forms of Cre recombinases, e.g., HNC, TCH6, HC, HNCM, CH. Incubation of fibroblast reporter cells with 1 μM HTNC for 1 to 2 hours can result in tranduction of 60 ~ 90% of the cells. Addition of 100 μM choroquine to culture medium can further enhance transduction and recombination.

Applications:

* In vitro LoxP recombination for subcloning or vector/clone engineering
* Transduction into cultured cells including stem cells ex Vivo

Enzyme Properties

Heat Inactivation: 40 units at 70°C for 10 minutes

Specific Activity: 10 units/μl

Purity: 95% by SDS-PAGE.

Reaction & Storage Conditions

Reaction Conditions: 1X Cre Recombinase Reaction Buffer, Incubate at 37°C.

1X Cre Recombinase Reaction Buffer:
50 mM Tris-HCl
33 mM NaCl
10 mM MgCl2
pH 7.5 @ 25°C

Unit Definition:

One unit is defined as the amount of enzyme necessary to produce maximal site-specific recombination of 0.25 μg LoxP control DNA in 30 minutes at 37°C in a total reaction volume of 50 μl. Maximal recombination is determined by agarose gel analysis and by transformation of reactions followed by selection on ampicillin plates.


Storage Conditions:
25 mM Tris-HCl
70 mM NaCl
10% Glycerol
pH 7.5 @ 25°C

Storage Temperature: -20°C or -80°C

In vitro Recombination Protocol

1. Prepare the Creator reaction mixture as follows:

400 ng Donor Vector (containing insert of interest)
400 ng Acceptor Vector
1 μL 10 X Cre Reaction Buffer
1 μL 10 X BSA (1 mg/ml)
2 μL Cre recombinase
dH20 up to 10 μl

A master mix of reaction buffer, BSA, ddH20, and Cre (and donor or acceptor if applicable) should be used.

2. Mix well by gently tapping the tube. Centrifuge briefly.

3. Incubate at room temperature for 15 minutes. Do not extent incubation past 15 minutes. Competing recombination reactions can reduce the yield of desired recombinants.

4. Incubate the tube at 70 ^oC for 5-10 minutes to stop the reaction.

5. Transform competent cells (> 1 X 10⁸ cfu/mg) with 1 ml of the reaction. Plate the transformation on an LB-agar plate containing 30 mg/ml chloramphenicol and 7% sucrose (w/v) to select for the correct recombinant vector. Allow the plates to dry for 10 minutes before inverting them.

6. Transforming into cells at 10⁷-10⁸ cfu/mg should yield about 5 to 10 colonies.

Transduction of Cre recombinase (HTNC) into cultured cells

1. Add choroquine (up to 100 μM) to cultured medium.
2. Add appropriate amount (1 to 10 μM) of HTNC to the medium and incunate uo to 24 hours. Note: serum-free medium can significantly increase transduction efficiency.
3. Change back to normal growth medium.
4. Determine transduction efficiency.