Sample Data Sheet
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1. Pre made tetracycline regulator TetR expression lentiviral particles user manual Product Name Amounts j 5 iviral CMV LVP017 Bsd RFP CMY TetR Bsd RFP Lentiviral C particles LVP017 Bsd CMV TetR Bsd Lentiviral particles LVP017 Neo CMV TetR Neo Lentiviral particles LVP017 Puro CMV TetR Puro Lentiviral particles CMV TetR Hygro Lentiviral LVFVUi s nygro p EF1a TetR GFP Bsd Lentiviral LVP459 GB particles in DMEM medium Bis 5 EF1a TetR RFP Bsd Lentiviral containing 10 FBS and 10x Ten particles polybrene 60ug ml LVP459 RP EPLER RFP Puro Lentiviral particles LVP459 Bsd EF1a TetR Bsd Lentiviral particles EF1a TetR Neo Lentiviral particles LVP459 Neo LVP459 Puro LVP017 RB PBS CMVsTetR Bsd RFP Lentiviral particles in PBS LVP017 Bsd PBS CMV rerR Bsd Lentiviral particles in PBS LVP017 Neo PBS CMV TetR Neo Lentiviral particles in PBS LVP017 Puro PBS CMV TetR Puro Lentiviral particles in PBS CMV TetR Hygro Lentiviral LVP017 Hygro PBS jweorzsie t8s particles in PBS 200ul 5 x 10 IFU mL LV g ivi in PBS LVVP459 GB PBS EFla TetR GFP Bsd Lentiviral particles in PBS LVP459 RB PBS EFla TetR Bsd RFP Lentiviral particles in PBS LVP459 RP PBS Ete retn RFP Puro Lentiviral particles in PBS LVP459 Bsd PBS EFTa resn Bsd Lentiviral particles in PBS AMSBIO www amsbio com info amsbio com EF1a TetR Puro Lentiviral particles AIA UK amp Rest of the Wor2. Method B co transduce TetR lentiviral particles and expression particles for tetracycline inducible expression 1 Plate cells in 0 5 ml of complete medium each well in 24 well plate incubated at 37 C for overnight 2 At the time of transduction the cell density should be around 50 confluency Thaw TetR lentiviral particles add 50ul 100ul of TetR particles into one well depending on cell type Note active dividing cells have higher transduction efficiency therefore less virus is needed Optionally add polybrene into medium at final concentration of 6ug ml Note polybrene can enhance transduction efficiency but may be toxic to some cell lines like some primary neuron cells incubate cells at 37 C for 24 hours 3 Then add 50ul of inducible expression particles into the well of 24 well plates Note ideally the TetR particles expression particles MOI ratio should be 5 1 but in general you should use more TetR particles than expression particles incubate at 37 C for overnight 4 Add tetracycline to induce expression The amount of tetracycline to use is dependent on cell type a common used final concentration is 1ug ml Note many bovine sera used in culture are contaminated with tetracycline or its derivatives which can affect basal expression 5 Alternatively at 48 72 hours after both transductions add antibiotics to select for stably transduced cells Note add both antibiotics for TetR particles and expression particle
3. cible expression of a human or mouse target Inducible GFP CAT LVP024 Control GFP RFP or YFP lentiviral expression YFP CAT LVP357 particles for validation of inducible control viruses RFP CAT LVP531 expression TetR expression Premade TetR Stable celllines Used for tetracycline inducible stable celllines in HEK293 cells with different expression of any constructs with TetR antibiotic selection markers binding sequence in their promoter CAT SC005 Safety Precaution Lentiviral particles have adopted the most advanced lentiviral safety features using the third generation vectors with self inactivation SIN 3 UTR and the premade lentivirus is replication incompetent However please use extra caution when using lentiviral particles Use the lentiviral particles in Bio safety Il cabinet Ware gloves at all times when handling Lentiviral particles Please refer to CDC and NIH s guidelines for more details regarding the safety issues References 1 Annu Rev Microbiol 1994 48 345 69 2 Microbiol Mol Biol Rev 2005 Jun 69 2 326 56 3 NIH Guidelines for Biosafety Considerations for Research with Lentiviral Vectors link 4 CDC guidelines for Lab Biosafety levels link AMSBIO www amsbio com info amsbio com AIA UK amp Rest of the World Ee North America m Germany Switzerland AM 184 Park Drive Milton Park 1035 Cambridge Street Bockenheimer Landstr 17 19 Centro Nord Sud 2E Abingdon OX14 4SE UK Cambridg
4. e MA 02141 60325 Frankfurt Main CH 6934 Bioggio Lugano T 44 0 1235 828 200 T 1 617 945 5033 or T 49 O 69 779099 T 41 0 91 604 55 22 F 44 O 1235 820 482 T 1 800 987 0985 F 49 O 69 13376880 F 41 0 91 605 17 85 F 1 617 945 8218
5. entration of the appropriate antibiotic that is required to kill your untransduced cells and replace medium containing antibiotic every 2 3 days until resistant colonies can be identified Note it takes 2 5 weeks depending on antibiotic type AMSBIO www amsbio com info amsbio com PS v4 Panie Milien Rik e E Einn imer Landstr 17 19 Contre Nese Sud F Aea es io ses SA Sea m soe Sra eos ore RBs a F 44 O 1235 820 482 T 1 800 987 0985 F 49 O 69 13376880 F 41 0 91 605 17 85 F 1 617 945 8218 5 Pick several resistant colonies and expand each clone into flask and assay for Tet repressor expression by ELISA or qRT PCR Note TetR assay materials need to be obtained and is not provided with TetR particles Alternatively you may pool the heterogeneous population of resistant cells and assay for Tet repressor expression If Bsd RFP dual marker TetR particles Cat LVP017 Bsd RFP are used the TetR expression cells can be identified via RFP fluorescence under microscope 6 Positive transduced cells are ready for transfection with inducible expression vectors or transduction with inducible expression lentiviral particles for the tetracycline inducible expression 7 At 48 hours after delivery target expression vector particles change medium and add tetracycline to induce expression of the target Note vary the final concentration of tetracycline from 0 01 ug ml to 1 ug ml to obtain different expression levels of target
6. hanced EF1a promoter The suCMV promoter provides highest TetR protein levels The enhanced EF1a promoter is non tissue specific promoter active in almost all cell types and is not silenced after long term cell culture Amsbio provides the TetR expression particles with different antibiotic selection markers or fluorescent antibiotic fusion dual markers under a separate RSV promoter Please see the vector map schemes below for the expression lentivector core structure RFP Bsd RFP Puro GFP Bsd Hygro WPRE Neo Puro Bsd VSV G pseudotyped lentiviral particles are generated in 293T cell and provided as 200ul per tube in 1 packaged in DMEM medium with 10 of FBS with 10x polybrene or 2 re suspended in PBS solution The virus in PBS is used to transduce the cells that do not tolerate serum and polybrene in the culture medium AMSBIO www amsbio com info amsbio com AIA UK amp Rest of the World Ee North America m Germany Switzerland AM 184 Park Drive Milton Park 1035 Cambridge Street Bockenheimer Landstr 17 19 Centro Nord Sud 2E Abingdon OX14 4SE UK Cambridge MA 02141 60325 Frankfurt Main CH 6934 Bioggio Lugano T 44 0 1235 828 200 T 1 617 945 5033 or T 49 O 69 779099 T 41 0 91 604 55 22 F 44 O 1235 820 482 T 1 800 987 0985 F 49 O 69 13376880 F 41 0 91 605 17 85 F 1 617 945 8218 TetR is used in tetracycline inducible expression It binds to any inducible promoters
7. ld gE North America m Germany Switzerland AM 184 Park Drive Milton Park 1035 Cambridge Street Bockenheimer Landstr 17 19 Centro Nord Sud 2E Abingdon OX14 4SE UK Cambridge MA 02141 60325 Frankfurt Main CH 6934 Bioggio Lugano T 44 0 1235 828 200 T 1 617 945 5033 or T 49 O 69 779099 T 41 0 91 604 55 22 F 44 O 1235 820 482 T 1 800 987 0985 F 49 O 69 13376880 F 41 0 91 605 17 85 F 1 617 945 8218 LVP459 Neo PBS EFla TetR Neo Lentiviral particles in PBS LVP459 Puro PBS EFla TetR Puro Lentiviral oo particles in PBS Storage lt 70 C avoid repeat freeze thaw cycles Stable for gt 6 months at lt 70 C Product Description Lentiviral system is a gene delivery tool using lentivectors for gene expression or knockdown Lentivectors are HIV 1 Human Immunodeficiency Virus 1 derived plasmids used to generate lentiviral particles lentivirus that can be transduced into almost all kinds of mammalian cell types or organs including stem cells primary cells and non dividing cells both in vivo and in cell culture system Particles can stably integrate into transduced cell s genome for long term expression Therefore lentivirus holds unique promise as gene transfer agents Pre made tetracycline repressor TetR lentiviral particles are generated from re engineered lentivector system Sequence verified TetR gene is constitutively expressed under either our proprietary super CMV promoter suCMV or an en
8. n particles and the double transduced cells will demonstrate a tetracycline dose dependent inducible expression of the target They can be co transduced with any inducible target expression lentiviral particles The double transduced cells selected via two antibiotic markers will demonstrate a tetracycline dose dependent inducible expression of the target Protocols as a general reference Method A transduce TetR lentiviral particles alone to generate TetR stable cell line 1 Plate cells in 0 5 ml of complete medium into each well of 24 well plate incubate at 37 C overnight 2 At the time of transduction the cell density should be around 50 confluent Thaw TetR lentiviral particle add 20ul 100ul into one well depending on cell types Note actively dividing cells have higher transduction efficiency therefore less virus is needed Optionally add polybrene into medium at final concentration of 6ug ml Note polybrene can enhance transduction efficiency but it may be toxic to some cell lines like some primary neuron cells incubate cells at 37 C for 72 hours 3 Remove the medium and replace with fresh complete medium containing the appropriate amount of antibiotic depending on TetR particle types to select for stably transduced cells 4 Trypsinize cells and pass into new well in 24 well plate in complete medium with appropriate amount of antibiotic Note a kill curve may have to be tested first to determine the minimal conc
9. rive Milton Park __ 1035 Cambridge Street Bockenheimer Landstr 17 19 Centro Nord Sud 2E Abingdon OX14 4SE UK Cambridge MA 02141 60325 Frankfurt Main CH 6934 Bioggio Lugano T 44 O 1235 828 200 T 1 617 945 5033 or T 49 O 69 779099 T 41 0 91 604 55 22 F 44 O 1235 820 482 T 1 800 987 0985 F 49 O 69 13376880 F 41 0 91 605 17 85 F 1 617 945 8218 Key features 1 High level of TetR expression driven by a super CMV promoter or an enhanced EF1a with demonstrated minimal basal expression from inducible expression vectors or their expression particles 2 Delivery of TetR expression lentivirus into dividing and non dividing host mammalian cell lines via high virus titers 3 Different antibiotic selection satisfies the requirement for different inducible expression vectors particles i e the double antibiotic selection 4 Depending on the cell lines and the TetR expression levels normally a 20 fold to 1000 fold induction can be obtained after addition of tetracycline Applications The premade TetR lentiviral particle is the best method to deliver the TetR protein Amsbio provides TetR expression lentiviral particles with different antibiotic markers TetR expression particles can be used as follows 1 2 They can be transduced alone into any host cell of your interest to generate TetR expression stable cell line The generated stable cell line is then transduced with any inducible target expressio
10. s at the same time to select double transduced cells 6 After obtaining the double transduced cells add tetracycline to induce expression of the gene of interest Note vary the concentration of tetracycline from 0 1 ug ml to 2 ug ml to obtain different expression levels of target Example Control for inducible expression The following picture demonstrated the GFP expression from premade inducible GFP lentiviral particles Cat LVP024 before and after induction AMSBIO www amsbio com info amsbio com Sy UK amp Rest of the World Ee North America m Germany Switzerland AM 184 Park Drive Milton Park 1035 Cambridge Street Bockenheimer Landstr 17 19 Centro Nord Sud 2E Abingdon OX14 4SE UK Cambridge MA 02141 60325 Frankfurt Main CH 6934 Bioggio Lugano T 44 0 1235 828 200 T 1 617 945 5033 or T 49 O 69 779099 T 41 0 91 604 55 22 F 44 O 1235 820 482 T 1 800 987 0985 F 49 O 69 13376880 F 41 0 91 605 17 85 F 1 617 945 8218 Left image Inducible GFP particles LVP024 transduced on HEK293 cells Middle image Inducible GFP particles transduced in TetR stable cells Right mage Inducible GFP particles transduced in TetR stable cells and induced by ug ml Tet Related products Applications Product series Premade expression ready e Used for constitutive expression gt 500 CAT lentiviral particle of human or mouse targets for human and mouse genes Used with TetR particles together for indu
11. that have incorporated Tet binding sequence to repress target expression Target expression is induced once tetracycline is added The added tetracycline binds to TetR which releases TetR from target s promoter and starts the transcription suCMV amp Eg Tet operator gene tetR protein Expression particles Expression repressed 1 tetR protein binds to tet operator sequences in promoter to repress the transcription l Add tet E Expression induced Tetracycline binds to tetR to release tetR from promoter and transcription is initiated Amsbio s lentiviral inducible expression vectors contain a strong constitutive promoter CMV or H1 that integrated with two copies of tetracycline regulator binding sequences This modification does not change promoter s constitutive expression status The GOI gene of interest can be expressed in high level as regular promoter without any induction However optionally lentiviral particles can be turned into tetracycline inducible system by using TetR expressing particles To achieve this inducible expression the TetR protein has to be present to bind block the expression in advance And the expression is induced after addition of tetracycline which removes the TetR from the promoter See the illustration figure below about the inducible mechanism AMSBIO www amsbio com info amsbio com AIA UK amp Rest of the World Ee North America Germany Switzerland ES 184 Park D
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