GenTarget`s EcoTMPlasmid DNA Miniprep Kit User Manual
Contents
1. 7930 Arjons Drive Suite B San Diego CA 92126 Phone 858 6788683 Email orders gentarget com Fax 800 3804198 GenTaruet Ine Pre made Expression Lentivirus for Monitoring CRE Recombination Reactions Catalog Product Name Description Number Pre made lentiviral LVP460 particles expressing LoxP GFP Stop Puro LoxP RFP Stop cassette under suCMV promoter It also contains a puromycin LVP460 nue Puro PBS antibiotic selection marker under Rsv 200ul per vial x _______ promoter 1x10 IFU ml in DMEM medium with Pre made lentiviral 10 FBS and 10x LVP460 particles expressing LoxP GFP Stop polybrene Neo LoxP RFP Stop cassette under suCMV amp LVP460 Neo PBS promoter It also contains a Neomycin Or antibiotic selection marker under Rsv promoter 200ul per vial x 5x10 IFU ml in Pre made lentiviral PBS solution LVP460 particles expressing LoxP GFP Stop Bsd LoxP RFP Stop cassette under suCMV amp promoter It also contains a Blasticidin Ee antibiotic selection marker under Rsv Bsa B gt promoter Storage lt 70 C avoid repeat freeze thaw cycles Stable for gt 6 months Product Description 1 Instruduction GenTarget s lentivector system is Human Immunodeficiency Virus 1 HIV based plasmids for gene expression and knockdown The lentivectors are used to generate lentiviral particles lentivirus that can be transduced into almost all kinds of mammalian cells inclu2. CRE recombination is not possible for all sites in every cell there will be many cells demonstrating both GFP and RFP signals after the addition of CRE recombinase The important observation is the dramatic increase in RFP positive cells following addition of CRE And the RFP GFP intensity ratio reflects the CRE recombination rate User manual Pre made lentivirus of LoxP GFP Stop LoxP RFP CRE reporting lentivirus Page 3 of 7 www gentarget com GenTarget Inc Copyrights 2015 7930 Arjons Drive Suite B San Diego CA 92126 Phone 858 6788683 Email orders gentarget com Fax 800 3804198 Gentarsot Ine Application protocol for reference only 1 Adhesive cells Transduction Protocols Note A quick transduction protocol is add 50ul virus into one well in 24 well plate where cell density is at 50 75 At 72 hours after virus added no need to change medium visualize the positive rate under fluorescent microscope For stable cell line generation pass cell into antibiotic containing medium or sort the cells via fluorescent signal Or simply select the cells by antibiotics Day 0 Seed the desired cells in complete medium at appropriate density incubate overnight Note at the time of transduction it grows to 50 75 confluent For example seed Hela cells at 0 5 x 10 ml x 0 5ml in a well of a 24 well plate Day 1 Remove the culture medium Add fresh warmed complete medium 0 5ml Thaw the Pre made lentivira
3. name to see product s pages Human Premade lentivirus expressin a human mouse or rat gene with mouse or rat RFP Blastididin fusion dual markers ORFs Fluorescent Preamde lentivirus express human codon optimized fluorescent markers protein GFP RFP CFP BFP YFP Luciferase Premade lentivirus for all kinds of luciferase protein expression expression firefly and Renilla with different antibiotic selection markers CRE Premade lentivirus for expressing nuclear permeant CRE recombinase recombinase with different flurescent and antibiotic markers LoxP Premade lentivirus expressing LoxP GFP Stop LoxP RFP ColorSwitch cassette used to monitor the CRE recombination event in vivo CRISPR hu Preamde lentivirus express humanzied wild type Cas9 CAS9 endonuclease for genomic editing with CRISPR TetR Premade lentivirus expressin TetR tetracycline regulator inducible protein the repressor protein for the inducible expression expression system repressor User manual Pre made lentivirus of LoxP GFP Stop LoxP RFP CRE reporting lentivirus Page 6 of 7 www gentarget com GenTarget Inc Copyrights 2015 7930 Arjons Drive Suite B San Diego CA 92126 Phone 858 6788683 Email orders gentarget com Fax 800 3804198 GenTaruet lnc Premde lentivirus for human and mouse iPS Myc NANOG iPS factors OCT4 SOX2 FLF4 factors with different fluorescent and antibitoic markers T antigen Express SV40 larg
4. Arjons Drive Suite B San Diego CA 92126 Phone 858 6788683 Email orders gentarget com Fax 800 3804198 GenTaruet lnc Safety Precaution Gentarget lentiviral particles adapts must advanced lentiviral safety features using the third generation vectors with self inactivation SIN 3UTR and the premade lentivirus is replication incompetent However please use extra caution when using lentiviral particles Use the lentiviral particles in Bio safety II cabinet Wear glove all the time when handling Lentiviral particles Please refer CDC and NIH s guidelines for more details regarding to safety issues References 1 Sauer B 1987 Functional expression of the Cre Lox site specific recombination system in the yeast Saccharomyces cerevisiae Mol Cell Biol 7 2087 2096 2 Stanislaw J Kaczmarczyk and Jeffrey E Green Nucleic Acids Res 2001 June 15 29 12 e56 Warranty This product is for research use only It is warranted to meet its quality as described when used in accordance with its instructions GenTarget disclaims any implied warranty of this product for particular application In no event shall GenTarget be liable for any incidental or consequential damages in connection with the products GenTarget s sole remedy for breach of this warranty should be at GenTarget s option to replace the products Related Products GenTarget s Pre made lentivirus Products Product Description Category please click category
5. ding stem cells primary cells and non dividing cells both in vivo and in vitro lLentiviral Particles stably integrate into the transduced cells genome for long term expression making it a great gene transfer agent CRE recombinase from bacteriophage P1 catalyzes recombination between 34 base pair target sequences called lox sites and can join individual plasmids User manual Pre made lentivirus of LoxP GFP Stop LoxP RFP CRE reporting lentivirus Page 1 of 7 www gentarget com GenTarget Inc Copyrights 2015 7930 Arjons Drive Suite B San Diego CA 92126 Phone 858 6788683 Email orders gentarget com Fax 800 3804198 GenTaruet lnc containing lox sites CRE recombination provides an excellent tool for conditional gene targeting in transgenic animal models by linking genotypic alterations to the biological outcomes phenotypes By inserting a LoxP flanked expression target into a host s genome target expression can be controlled via CRE recombinase Expression of LoxP flanked target genes may occur prior to the addition of CRE enzyme when CRE is applied it deletes the LoxP flanked target segment and stops the target expression Simultaneously CRE mediated recombination can activate expression of a second target downstream from the deleted segment GenTarget provides pre made CRE reporting lentivirus for easy fast and convenient testing and monitoring of CRE recombination efficiency in vivo and in vitro This l
6. e T antigen with different selection markers Expression Cell Premade lentivirus for cell organelle imaging The fluorescent Organelle marker GFP RFP CFP was sub cellular localized in different imaging cell organelle for living cell imaging LacZ Express different full length B galactosidase lacZ with different expression selection markers Anti miNA Pre made lentivirus expression a specific anti miRNA cassette lentivirus Fluorescent Pre made lentivirus expression a GFP RFP CFP ORF fusion ORF fusion target Pre made Premade shRNA lentivirus for knockdown a specific genes P53 shRNA LacZ Luciferase and more lentivirus microRNA Premade lentivirus expression human or mouse precursor and anti miRNA And anti miRNA lentivector and virus for human and microRNA mouse miRNA lentivirus Negative Premade negative control lentivirus with different markers control serves as the negative control of lentivurs treatment for lentiviruses validation of the specificity of any lentivirus target expression effects Ready to use lentivirus expressing specific enzymes with different selection markers expression User manual Pre made lentivirus of LoxP GFP Stop LoxP RFP CRE reporting lentivirus Page 7 of 7 www gentarget com GenTarget Inc Copyrights 2015
7. entivirus has been engineered to constitutively express the LoxP GFP stop LoxP RFP Stop cassette under a super CMV promoter See the expression cassette scheme below It reports the occurrence of CRE mediated recombination events via a color switch mechanism thereby providing an esay fast and continual monitoring for the presence of CRE or CRE recombination event Without CRE enzyme ATG cmv Pro PG gt With CRE express 2 Multiple Selection Markers GenTarget offers the CRE reporting lentivirus with a broad variety of antibiotic selection markers including puromycin neomycin or blasticidin The selection markers are expressed under an RSV promoter not shown in the schematic above User manual Pre made lentivirus of LoxP GFP Stop LoxP RFP CRE reporting lentivirus Page 2 of 7 www gentarget com GenTarget Inc Copyrights 2015 7930 Arjons Drive Suite B San Diego CA 92126 Phone 858 6788683 Email orders gentarget com Fax 800 3804198 GenTaruet lnc These markers allow easy selection for transduction positive cells those expressing the CRE detection cassette by antibiotic resistance Two Formats The LoxP ColorSwtich particles are provided as at 200 pl vial in two formats e DMEM medium with 10 FBS and 60 ug ml polybrene 10x e PBS solution which is best for in vivo applications cell cultures requiring serum free conditions or hard to infect cells How ColorSwtic
8. h Reporting Works The CRE reporting lentivirus is used to monitor or confirm the efficiency of CRE recombination jin vivo It is an easy and very effective tool for verifying the performance of CRE loxP systems CRE expression plasmids pre made CRE lentiviral particles and purified CRE enzyme in vivo and can serve as a control indicator for the same systems The CRE reporting lentivirus demonstrates a strong GFP fluorescence after infection into mammalian cells but does not show an RFP fluorescence signal Once the CRE protein is present in the nucleus CRE excises deletes the DNA fragment between two loxP sites As a result the GFP is removed and RFP is expressed with a resulting switch to RFP fluorescence The ratio of RFP expressing to GFP expressing cells can be easily monitored via fluorescence cell sorting visualized by microscopy or the fluorescence intensity may be measured by a fluorometer with GFP and RFP filter sets See the sample results below Notes Like any mammalian pol II promoter the CMV promoter could seek any possible ORFs and in some cell types it may slightly express the 2nd ORF the RFP in this case which is considered the basal or leaking RFP signal Tt is not possible for CRE to be delivered into all cells therefore some GFP positive cells may still be detected even after the application of CRE Also because some cells may integrate multiple copies of the LoxP GFP LoxP RFP cassette and since 100
9. haking in CO2 incubator At 24 hours after transduction add equal amount of fresh medium containing related antibiotics Note each particles contain an antibiotic marker and the antibiotic amounts to use depends upon cell types Grow cell in CO incubator At 72 hours after transduction check fluorescence under microscope or calculate the transduction efficiency using cell sorting machine like FACS or Guava machine You can sort the fluorescent positive cells and maintain the antibiotic selection to generate stable cell lines Note GFP filter wavelength Ex450 490 Em525 RFP filter Ex545 Em620 Sample images of CRE loxP recombination detection Without CRE With CRE GFP filter RFP filter GFP filter RFP filter Left panel without CRE CRE reporter cell line Cat SCO18 Bsd was created by LoxP460 Neo particles cultured in a 24 well plate Images were taken with a GFP filter set Ex 490nm Em 525nm and an RFP filter set Ex 545nm Em 620nm Right panel with CRE CRE reporter cell line Cat SCO18 Bsd was created by LoxP460 Neo particles cultured in completed in 24 well plate 50 ul of CRE expression lentiviral particle Cat LVP339 was added into the cells in one well Images were taken at 72 hours after the addition of CRE expression lentivirus User manual Pre made lentivirus of LoxP GFP Stop LoxP RFP CRE reporting lentivirus Page 5 of 7 www gentarget com GenTarget Inc Copyrights 2015 7930
10. l stock at room temperature Add appropriate amount of virus stock to obtain the desired MOI Return cells to 37 C CO incubator Try to avoid thaw and freeze cycles for pre made lentivirus But if you cannot use all virus in one time you still can re freeze the virus at 800C for future use But virus titer will decrease by 10 for each re thaw Day 3 At 72hr after transduction check the transduction rate via fluorescence image with a suitable filter under fluorescent microscope or calculate the exact transduction rate via Flow Cytometry System FACS or any flow cytometry such as Guava machine Note You should only see GFP signal at this stage before you apply CRE enzyme to the cells Day 3 Transduced cells can be sorted out via FACS selected by its specific antibiotics A pilot experiment should be done to determine the antibiotic s kill curve for your specific cell line Refer to any literatures about How to generate stable cell lines CRE enzyme delivery The selected cell should demonstrate strong GFP signal and should have no RFP signal After the cell selection the cells are ready used as an indicator cell line for CRE recombination activity e Apply the CRE enzyme into the cells which can be achieved by infected cell with CRE expression lentivirus or by regular lipid transfection of a CRE expression plasmid or even simply by adding purified penetrating CRE protein enzyme Note Gentarget provides ready to use CRE ex
11. pression lentivirus with different antibiotic selection marker for CRE delivery into cells e Put cells in normal culture conditions for 48 72 hours e Detect CRE recombination reaction The RFP signal will gradually showed up and peaked at 48 hours or longer times dependent upon CRE delivery methods after the CRE delivery The RFP GFP cell population ratio or the RFP signal intensity reflects the CRE LoxP recombination efficiency rate You can sort the cell by FACS machine other meters or visualize the RFP positive cell under fluorescent signal User manual Pre made lentivirus of LoxP GFP Stop LoxP RFP CRE reporting lentivirus Page 4 of 7 www gentarget com GenTarget Inc Copyrights 2015 7930 Arjons Drive Suite B San Diego CA 92126 Phone 858 6788683 Email orders gentarget com Fax 800 3804198 Gentarsot Ine 2 Suspension cells transduction Protocols 1 PA 3 Grow your cell in your completed suspension culture medium shaking in flask in CO incubator if necessary Measure cell density When cell grow to 3 x 10 cell ml measure cell viability should be gt 90 then diluted cells into 1 x 10 cell ml in completed medium Transduction thaw lentiviral particles at room temperature Simply add premade lentiviral particle into the diluted cells at ratio of 50 to 100ul virus per 0 5 ml of cells Note depending on the cell types you may need to use more or less viruses Grow cells in flask s
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