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Genome Engineering Enzymes

iCP-Cre

improved Cell-Permeable Cre recombinase

T. Amount Volume Package Size Concentration Price Qty Cart Buy Now
3 mg 1 ml X 2 ea 2 ml Cap Tube (小) 1.5 μg/μl $ 500.00
5 mg 1 ml X 2 ea 2 ml Cap Tube (小) 1.5 μg/μl $ 800.00
Description

The Cre recombinase from bacteriophage P1 has been widely used to induce DNA sequence-specific recombination in mammalian cells. Applications involving Cre-LoxP recombination have included conditional mutagenesis, gene replacement, chromosome engineering in mice and conditional gene expression. 

CELLIVERY has developed a cell-permeable DNA recombinase enabling penetration into cell membrane for epigenetic regulation of gene structure and function with improved cell-permeable iCP-Cre recombinase, named iCP-Cre. iCP-Cre exploits the novel protein-based delivery system for genetic modification of the growing list of mice with conditional alleles. iCP-Cre will facilitate gene-function studies and provide useful tool for various fields of scientists.

Figure 1. Benefits of iCP-Cre

  • iCP-Cre is a cell permeable recombinant protein which takes an advantage of our proprietary technology called Therapeuticmolecule Systemic Delivery Technology. The technology allows Cre recombinase to permeate through the cell surface membrane and nuclear membrane, allowing genome engineering to be done efficiently and effectively.
  • iCP-Cre can knock out loxP sites tissue specifically with high efficiency.
  • Genome engineered animal model can be produced from loxP floxed mouse in a short period of time.
  • Reduced cost for mouse breeding
  • Quantitative delivery of Cre recombinase is possible in real time.
  • Homogeneous epigenetic genome engineering within the tissue prevents imbalanced phenotype expression.
  • iCP-Cre can even penetrate blood-brain barrier effectively.
  • By using many alternative administration routes of iCP-Cre, such as direct injection, nasal injection and catheter delivery, organ/tissue-specific genetically engineered animal models are possible.
Properties
  • Cat. No : CV09~
  • Expression Host : E.Coli
  • Concentration : 1.5 ㎍/㎕
  • Purity : >95% by SDS PAGE
  • Endotoxin : <100 EU/mg
  • Specific Activity : 5 X 106 CFU/mg of protein
  • Storage Buffer : 50 mM Tris-HCl, pH 9.0, 150 mM NaCl, 10% Glucose
  • Storage Temperature : -80℃
FAQ / Q&A

How is systemic and organ-specific recombination achieved with iCP-Cre?

Answer : Systemic and organ-specific recombination is done with same iCP-Cre recombinant protein. The only difference lies in the administration route. For the systemic recombination, iCP-Cre is administered by IV whereas organ-specific recombination is administered locally.

What is the molecular weight of iCP-Cre?

Answer : 73 kDa

What are the possible administration routes for iCP-Cre?

Answer : iCP-Cre is administered by IV, which flow along the blood stream and enter into blood cells (PBMCs), then distribute to the entire body via blood vessel network. The iCP-Cre in blood stream (blood plasma and blood cells) can penetrate into endothelial cells of blood vessels and deliver systemically into cells and tissues in vivo. 
iCP-Cre is systemically distributed in vivo by the ability of aMTD-mediated cell-to-cell delivery. However, if the protein is administered subcutaneously (SC), distribution is restricted locally according to the amount of the protein applied.

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Reference
  • 1. Jo et al. (2001) Nature Biotechnology, 19(10):929-933

    Download
  • 2. Lin et al. (2004) BMC Biotechnology, 4(25):1-13

    Download
  • 3. Jo et al. (2005) Nature Medicine, 11(8):892-898

    Download
  • 4. Jeon et al. (2010) Nature Neuroscience, 13(4):482-488

    Download
Cautions

· Always store in deep freezer (under - 80oC).
· Always thaw iCP-Cre on ice before use and keep it over the ice during the experiments.
· Avoid freeze/thaw cycle.
· Remember that to produce genetically modified animals (ex. KO or KI mouse) with iCP-Cre, one (1) vial is needed to be injected over five (5) consecutive days. Therefore, aliquot appropriate amounts to prevent freeze/thaw repetition.

Protocol I: Whole-body Recombination

1. Intravenous (IV) inject 200 μl of iCP-Cre (1.5 μg/μl) into any floxed mouse’s tail vein every day for five (5) consecutive days.
2. Allow two-to-three (2-3) days after the fifth (5th) injection before analyzing the recombination and phenotype.

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NOTE When administering via tail vein, it is important to start from the end of the tail and ascend towards the base of the tail with each injection (Figure 2). This is to avoid any leakage due to tissue damage endured by the previous injection.



Figure 2. IV Injections for Systemic (Whole-Body) Recombination


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Figure 3. In Vivo Recombination Activity of iCP-CreROSA26-LSL-lacZ and ROSA26-LSL-EYFP are transgenic mice lines in which Cre-mediated recombination activates expression of β-galactosidase reporter gene and yellow fluorescence protein (YFP) reporter gene, respectively.



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Figure 4. Systemic Recombination with iCP-Cre in ROSA26-LSL-LacZ Mouse. High levels of recombination are observed in all tissues examined after tail vein injection of iCP-Parkin, including the brain as evidenced by intense blue staining of intact organs. This is a direct evidence of BBB crossing by iCP-Cre. 




Protocol II: Organ-specific Recombination

Recombination Target Organ

Administration

Route

Dose

Injection Term

(# of injections)

Sacrifice

(from the last injection)

Brain

Cisterna

Magna

3.6 mg/kg

(90 mg/head)

Once

2 days later

1.2 mg/kg

(30 mg/head)

Total five, once every two days

(10 days total)

2 days later

Liver

Portal vein

4.0 mg/kg

(100 mg/head)

Once

7 days later

Kidney

Intrarenal

4.0-6.0 mg/kg

(100-150 mg/head)

Once

7 days later


Table 1. Protocols for Organ-Specific Recombination of iCP-Cre



Brain-Specific Recombination

1. Administer iCP-Cre (3.6 mg/kg/day or 1.2 mg/kg/day) to any floxed mouse by cisterna injection for 1 or 5 days depending on the injection dosage.
2. Allow 2 days after the last injection before analyzing the recombination and phenotype.


Liver-Specific Recombination

1. Administer iCP-Cre (4.0 mg/kg/day) to any floxed mouse by portal vein injection for 1 day.
2. Allow 7 days after the last injection before analyzing the recombination and phenotype.


Kidney-Specific Recombination

1. Administer iCP-Cre (4.0-6.0 mg/kg/day) to any floxed mouse by intrarenal injection for 1 day.
2. Allow 7 days after the last injection before analyzing the recombination and phenotype.


Protocol III: In Vitro Recombination

1. Plate cells (1X105 cells/well,12 well) in culture dish.
2. Add appropriate amount (2 μM) of iCP-Cre to the serum free medium (RPMI1640) and incubate for 2 hours. Note : Serum inhibits transduction of iCP-Cre
3. Wash the cells with PBS 2 times.
4. Add growth medium.
5. After 24 ~ 48 hours, determine recombination efficiency.


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Figure 5. In Vitro Recombination Activity of iCP-Cre. To investigate the biological activity of the iCP-Cre, color-switch reporter cells containing LoxP sites were used. The LoxP-DsRed-LoxP-eGFP cell-line was used for this experiment, it has green fluorescent protein (GFP) reporter gene that only becomes activated with successful Cre recombination. The cells were treated with 1 μM of the iCP-Cre for 2 hours at 37oC. GFP expression level was measured after 24 hours using fluorescence microscopy and confirmed that the iCP-Cre recombination has successfully occurred.

PDF File
  • 1. Jo et al. (2001) Nature Biotechnology, 19(10):929-933

    Download
  • 2. Lin et al. (2004) BMC Biotechnology, 4(25):1-13

    Download
  • 3. Jo et al. (2005) Nature Medicine, 11(8):892-898

    Download
  • 4. Jeon et al. (2010) Nature Neuroscience, 13(4):482-488

    Download
Quality Control Assays

The following Quality Control Tests are performed on each new lot to check if they meet the specifications designated for the product:

· SDS PAGE · Recombination Activity (Colony Formation assay)
· The circular substrate containing LoxP sites and Ampicillin resistance gene were constructed in pET-28a(+) vector. 0.2 μg of iCP-Cre was incubated with 150 μg of the substrate for 30 minutes at 37oC in 50 μl of reaction buffer (33 mM NaCl, 50 mM Tris-HCl and 10 nm  M MgCl2). The mixture was incubated at 70oC for 10 minutes for inactivation, and was left on ice for 5 minutes. The mixture was then transformed into E. coli, and then, the colonies were formation was observed.
· Individual lot data can be found on the Product Summary Sheet below:

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Figure 6. Scheme of iCP-Cre’s recombination activity.

PDF File





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