Cre/loxP System,

lentiviral and retroviral particles for introducing Cre and loxP expression into target cells.

he bacterial phage P1 Cre/lox recombination system mediates DNA excision, integration, inversion and translocation in a sequence spe-

Box 1 | Product List

pTYF-loxP-nlCre-2AGFP-loxP pTYF-nlCre pTYF-nlCre-RFP fusion pTYF-nICre-2A-GFP loxP-RFP ABP-RP-Cre2AGPL ABP-RP-Cre2AGPLH ABP-RP-TLCCreL ABP-RP-TLCCreLH ABP-RP-CreRFPF ABP-RP-CreRFPFH ABP-RP-Cre2AGL ABP-RP-Cre2AGLH ABP-RP-TLCRFPLOXL ABP-RP-TLCRFPLOXLH 5 vials, 107TU in 0.1ml / vial 5 vials, 109TU in 10 l / vial 5 vials, 107TU in 0.1ml / vial 5 vials, 109TU in 10 l / vial 5 vials, 107TU in 0.1ml / vial 5 vials, 109TU in 10 l / vial 5 vials, 107TU in 0.1ml / vial 5 vials, 109TU in 10 l / vial 5 vials, 107TU in 0.1ml / vial 5 vials, 109TU in 10 l / vial

components: the 38-kDa Cre (cyclization recombination) recombinase and two 34 bp loxP (locus of X-over of P1) tion has been frequently applied for Viral vectors are widely used vehicles to bring genetic material inside eukaryotic cells. Compared to transfection methods such as cationic lipids, electroporation, or microinjection, induction with viral if an appropriate viral system with correct tropism is selected. In most cases, either adenovirus or lentivirus is used for introducing Cre to cells. Advantages and disadvantages comparing the two systems: 1.) Cre-expressing adenovirus is typically used high concentration (measured by titer of the viral stock and MOI during infection), which may cause toxicity to the cells; 2.) Cre-expressing lentivirus can be used at lower MOI for achieving similar effect as high doses of adenovirus, which promises a lower cell toxicity; 3.) Adenovirus does not integrate into the host genome, whereas lentivirus does. Allele Biotech provides custom retrovirus or lentivirus packaging services using proprietary technologies that are unique and 8 to 109 TU/ml, without any concentrating steps (US patents 6,207,455 and 6,531,123). These technologies along with optimal operation procedures enable Allele Biotech to package viruses at < 1% of the market price in certain categories on per million particle basis. Based on this strong platform, Allele Biotech now offers-

H denotes High Titer

pre-packaged, titer-determined, high titer lentiviral particles ready to infect target cells for Cre/loxP system. In this system, 3 product lines are available: 1) Lentiviral particles encoding mammalian cell-adapted Cre gene with a nuclear localization signal (nlCre) are among the viral gene expression tools offered by Allele Biotech. These SIN Lentiviral vectors behind the EF1 promoter. An option is available for having GFP as a reporter following Cre, separated by a 2A peptide if needed. Please review Fig 1. for more options. 2) Gryphon-based retroviral particles encoding nCre, with IRES signal and GFP for tracking expression. 3) Cre/loxP Reporter Lentiviral particles, containing loxP sites GFP as tracking markers. the Cre/loxP system also works well as a sensitive and convenient method for lentivirus titer determination.

or Research Use Only. Not for Diagnostic or Therapeutic Use. Purchase does not include or carry any right to resell or transfer this product either as a stand-alone product or as a component of another product. Any use of this product other than the permitted use without the express written authorization of Allele Biotech is strictly prohibited

Lentiviral vectors should be handled using NIH BSL-2 safety guidelines. For more information, please see Biosafety in Microbiological and Biomedical Laboratories (4th edition) which is available on the Web sites of the National Institutes of Health at: http://bmbl.od.nih.gov

Safety Information

Website: www.allelebiotech.com
Call: 1-800-991-RNAi/858-587-6645

Email: oligo@allelebiotech.com For Technical Support: Email:vivec@allelebiotech.com

Allele Biotech-Introducing Cost Effectiveness to Research

Protocols
MOI Determination with GFP Control Lentivirus: Storage: -80C. Avoid repeated freeze thaw cycles Steps: 1. plate before infection. 2. Place GFP-virus stock (virus titer: 1x108 IU/ml) in a 37C water bath and before totally thaw transfer onto ice immediately. 3. Make a serial dilution of 3-4 different multiplicity of infection (MOI = infectious units/ cell number) to test the target cells. e.g. 1x104 TE671 cells in each well of a 12-well plate: Table 1 | MOI Culture Medium (ul): Polybrene (2mg/ml): Virus (ul): Total volume (ul):
10MOI 20MOI 30MOI 60MOI

MOI Determination with loxP-FP Reporter Lentivirus : Storage: -80C. Avoid repeated freeze thaw cycles Steps: 1. plate before infection. 2. Place Cre Expression virus stock and loxPFP(GFP or RFP) reporter virus stock (virus titer: 1x108 IU/ ml) in a 37C water bath and before totally thaw transfer onto ice immediately. 3. Mix different volumes of Cre virus stock and loxP-FP reporter virus stock with growth media containing polybrene, as described in Table 1. 4. Tilt the plate gently every 30 min. After 3hrs, add 1 ml fresh medium. 5. After 48-72 hrs, evaluate Cre recombinase cytometry (loxP-RFP or GFP) . 6. toxicity.

198 1 1 200

197 1 2 200

195 1 4 200

189 1 10 200

4. Tilt the plate gently every 30 min. After 3hrs, add 1 ml fresh medium. 5. After 48-72 hrs, evaluate GFP expression by 6. toxicity. Figure 1 | Construct Detials

nICre nICre-2A-GFP

nICre

loxP

loxP

Allele Biotech-Introducing Cost Effectiveness to Research