RetroNectin® Recombinant Human Fibronectin Fragment
Contents
1. 3 Dissociate adherent cells from the plate with Cell Dissociation Buffer Life Technologies an enzyme free solution or trypsin EDTA following the manufacturer s instructions Note For many cell types adherent cells may be collected by pipetting only 4 Combine the cells obtained from steps 1 3 in the same tube and centrifuge to recover the cells 5 Wash the cells with HBSS Hepes twice collecting the cells by centrifugation Suspend the cells in HBSS Hepes for further analysis Any buffer or medium suitable for downstream application of the cells can be also used for resuspension EE TAKARA BIO INC URL http www takara bio com RetroNectin Cat T100A B Takaka Recombinant Human Fibronectin Fragment v201303 VI References 1 ia oS a Kimizuka F Taguchi Y Ohdate Y Kawase Y Shimojo T Hashino K Kato Sekiguchi K and Titani K 1991 Production and characterization of functional domains of human fibronectin expressed in Escherichia coli J Biochem 110 284 291 Hanenberg H Xiao XL Dilloo D Hashino K Kato and Williams DA 1996 Colocalization of retrovirus and target cells on specific fibronectin fragments increases genetic transduction of mammalian cells Mat Med 2 876 882 Hanenberg H Hashino K Konishi H Hock RA Kato I and Williams DA 1997 Optimization of fibronectin assisted retroviral gene transfer into human CD34 hematopoietic cells Hum Gene Ther 8 2193 22. 320 u l cm2 In this method some infection inhibitory molecules may not be removed by washing with PBS For this reason the efficiency of gene transduction might be reduced In such a case it is recommended that the virus stock solution be used after dilution with growth medium Optimization is required to determine the suitable dilution rate 2 Place the plate in a centrifuge pre warmed to 32 C and centrifuge for 2 hours at 32 C at 1 000 2 000X g to facilitate binding of virus particles with RetroNectin reagent 3 Discard the supernatant but do not allow the plate to dry Wash the plate with an appropriate volume of PBS or PBS containing 0 1 2 albumin BSA or HSA Then perform virus infection according to A 3 E TAKARA BIO INC URL http www takara bio com RetroNectin Cat T100A B Takaka Recombinant Human Fibronectin Fragment v201303 A 3 Virus Infection Prepare the target cells while the retrovirus particles are binding to the RetroNectin reagent coated plate It is important that the target cells be in logarithmic growth phase and express integrin receptors VLA 4 and or VLA 5 When using hematopoietic stem cells pre stimulation with cytokine may be necessary The cytokine type should be determined based on your specific research protocols Examples are cited in references 3 and 5 1 Collect the target cells and count the number of living cells Then suspend the cells in the growth medium at a concentration of 0
3. 2 1 x 105 cells ml 2 Remove the wash solution from the virus bound plates prepared by A 1 or A 2 Do not allow the plate to dry Immediately add target cells at a density of 0 5 2 5 x 104 cells cm2 Although the optimal cell density depends on cell size and growth rate the initial cell density should allow the cells to be actively growing or nearly confluent when analyzed 2 3 days after transduction When infecting more cells you may increase the cell density but the cells will need to be subcultured after gene transduction Note To promote contact between the target cells and viral particles plates can be centrifuged after adding the cells 3 Incubate in a 37 C 5 CO2 incubator for 2 3 days 4 Collect both non adherent and adherent cells 1 Transfer the supernatant to a centrifuge tube 2 Recover remaining non adherent cells by washing the plate with PBS 3 Dissociate adherent cells from the plate with Cell Dissociation Buffer Life Technologies an enzyme free solution or trypsin EDTA following the manufacturer s instructions Note For many cell types adherent cells may be collected by pipetting only 4 Combine the cells obtained from steps 1 3 in the same tube and centrifuge to recover the cells 5 Wash the cells with HBSS Hepes twice collecting the cells by centrifugation Suspend the cells in HBSS Hepes for further analysis Any buffer or medium suitable for downstream application of the ce
4. Hepes or PBS After removing the wash solution the plate is ready for use The RetroNectin coated plate can be sealed with Parafilm and stored at 4 C for up to one week 2 Gene Transduction There are two methods of gene transduction using RetroNectin reagent RetroNectin bound virus RBV infection method Section A and supernatant SN infection method Section B In the RBV infection method retroviral particles are first bound to the plate coated with RetroNectin reagent and the target cells are added after removing the virus supernatant In the SN infection method the virus solution and target cells are mixed and then added to the RetroNectin coated plate When virus solution is directly used for virus infection without purification the gene transduction efficiency may be reduced because of contaminating molecules that inhibit infection In such cases the RBV infection method is recommended With this method inhibitory molecules can be removed by binding viruses to RetroNectin reagent and removing the supernatant Note Using a transient production system such as the Retrovirus Packaging Kit Eco Ampho Cat 6160 6161 and the pDON AI 2 vector Cat 3654 3653 retroviral vectors can be prepared in one week Not available in all geographic locations Check for availablity in your region URL http www takara bio com TAKARA BIO INC RetroNectin Cat T100A B Takaka Recombinant Human Fibronectin Fragment v201303 A
5. Rasa N RGDS CS 1 4 L Ji Cell Heparin Li 4 I nO Le BH fs Le 1 I COOH hemo A TH TI lk t Cuz kurk Ll Fibrin Collagen DNA Cell Heparin Cell Fibrin Heparin III CS Blue binding domain Figure 1 The hypothesized mechanism of RetroNectin mediated transduction The cell binds to the CS 1 site via VLA 4 and to the C domain via VLA 5 The viral particle can bind to the H domain of RetroNectin These interactions increase the localized concentrations of cells and viral particles an effect that is thought to enhance gene transduction URL http www takara bio com TAKARA BIO INC RetroNectin Cat T100A B Takaka Recombinant Human Fibronectin Fragment v201303 Il Components RetroNectin Cat T100A 0 5 mg 0 5 ml RetroNectin Cat T100B 2 5 mg 2 5 ml Note RetroNectin is provided as a sterile 1 mg ml solution lll Storage 20 C Caution Freezing and thawing can be repeated up to 10 times Do not mix the solution vigorously Do not vortex IV Materials Required but not Provided Equipment Non treated tissue culture plates or dishes e Electric pipetter e Pipetter e Sterile pipettes e Sterile tips with filters e Safety cabinet or clean workstation e Microscope e CO2 incubator e Microplate centrifuge Reagents e Sterile PBS Phosphate Buffered Saline e HBSS Hepes Hank s Balanced Salt Solution supplemented with 2 5 v v 1 M Hepes e 2 BSA BSA Fraction V PBS Solution TAKARA BIO IN
6. RetroNectin Bound Virus RBV Infection Method A 1 Preparation of virus bound plate without centrifugation 1 Add retrovirus supernatant at 125 250 u l cm2 to a RetroNectin coated plate or dish 2 Incubate for 4 to 6 hours at 32 C or 37 C in a 5 CO2 incubator to promote binding of the virus particles with RetroNectin reagent 3 Discard the supernatant but do not allow the plate to dry Wash the plate with an appropriate volume of PBS or PBS containing 0 1 2 albumin BSA or HSA After washing perform infection according to A 3 A 2 Preparation of virus binding plate by centrifugation If the virus titer is high enough binding of viruses with RetroNectin reagent can be accomplished without centrifugation as described in A 1 However if the titer is low or if you require higher gene transduction efficiency binding the virus by centrifugation is preferable With this method the time required to bind the retrovirus is significantly less 2 hours versus 4 6 hours for the non centrifugation method A plate such as a non treated cell culture plate that can tolerate centrifugation at 1 000 2 000X g for 2 hours at 32 C is required for this method In addition please note there is a possibility of aerosol formation 1 Add the retrovirus stock solution or diluted solution at 125 500 pl cm2 to the RetroNectin coated plate Note The volume that can be added to the plate varies For a 6 well plate the upper limit is 3 ml
7. licenses for other use please contact us by phone at 81 77 543 7247 or from our website at www takara bio com Your use of this product is also subject to compliance with any applicable licensing requirements described on the product web page It is your responsibility to review understand and adhere to any restrictions imposed by such statements All trademarks are the property of their respective owners Certain trademarks may not be registered in all jurisdictions Pa TAKARA BIO INC URL http www takara bio com
8. 206 Pollok KE Hanenberg H Noblitt TW Schroeder WL Kato Emanuel D and Williams DA 1998 High efficiency gene transfer into normal and adenosine deaminase deficient T lymphocytes is mediated by transduction on recombinant fibronectin fragments J Virol 72 4882 4892 Chono H Yoshioka H Ueno M and Kato 2001 Removal of inhibitory substance with recombinant fibronectin CH 296 plates enhances the retroviral transduction efficiency of CD34 CD38 bone marrow cells J Biochem 130 331 334 VII Related Products Retroviral vectors pDON AI 2 Neo DNA pDON AI 2 DNA Cat 3653 Cat 3654 Preparation of Recombinant Retroviral Particles Cat 6161 Cat 6160 Clontech Cat 631508 Clontech Cat 631530 Retrovirus Packaging Kit Ampho Retrovirus Packaging Kit Eco Retro X System Retro X Universal Packaging System aS m n ee Lentiviral Vectors and Vector Systems Lenti X Expression System Lenti X Expression System EF 1alpha Version Preparation of Recombinant Lentiviral Particles Lenti X 293T Cell Line Lenti X HTX Packaging System Lenti X HTX Ecotropic Packaging System Other RetroNectin Pre coated Dish RetroNectin GMP Retrovirus Titer Set for Real Time PCR Retro X qRT PCR Titration Kit Lenti X qRT PCR Titration Kit Clontech Cat 632164 Clontech Cat 631253 Clontech Cat 632180 Clontech Cat 631247 Clontech Cat 631251 Cat
9. C URL http www takara bio com RetroNectin Cat T100A B Takaka Recombinant Human Fibronectin Fragment v201303 V Protocol 1 Preparation of RetroNectin Coated Plates Coat a plate using 20 100 u g ml RetroNectin with a volume corresponding to 4 20 ug cm plate area 1 Prior to coating prepare a RetroNectin solution 20 100 ug ml 1 by diluting with sterile PBS 1 Example of calculating amount of RetroNectin reagent When 2 25 ml of RetroNectin solution at a concentration of 20 ug ml is placed into a 35 mm diameter dish 9 cm2 the concentration used for coating is 5 ug cm2 Note To avoid loss of RetroNectin fragment do not filter sterilize RetroNectin solution diluted with PBS 2 Dispense an appropriate volume 2 of sterile RetroNectin solution into each plate and allow the plate to stand for 2 hours at room temperature or at 4 C overnight x2 Dispense 0 5 ml into each well of a 24 well plate or 2 ml into each well of a 6 well plate Note Non treated cell culture grade tissue culture plates or dishes should be used in this step 3 Remove the RetroNectin solution and then block with an appropriate volume 3 of sterile 2 bovine serum albumin BSA Fraction V in PBS Allow the plate to stand at room temperature for 30 minutes x3 Use 0 5 ml for each well of a 24 well plate or 2 ml for each well of a 6 well plate 4 Remove the BSA solution and wash the plate once with an appropriate volume of HBSS
10. T110A Cat 17201 Cat 6166 Clontech Cat 631453 Clontech Cat 631235 Not available in all geographic locations Check for availability in your region URL http www takara bio com TAKARA BIO INC E RetroNectin Cat T100A B Takaka Recombinant Human Fibronectin Fragment v201303 0S TAKARA BIO INC URL http www takara bio com RetroNectin Cat T100A B Takaka Recombinant Human Fibronectin Fragment v201303 NOTICE TO PURCHASER LIMITED LICENSE L9 RetroNectin A method to increase the efficiency of retrovirus mediated gene transfer covered by the claims of U S Patent No 5 686 278 6 033 907 7 083 979 and 6 670 177 and their foreign counterpart patent claims is licensed to TAKARA BIO INC exclusively and worldwide M69 RetroNectin Expansion Method This product is covered by the claims of Japanese Patent No 4406566 and its foreign counterpart patent claims URL http www takara bio com TAKARA BIO INC RetroNectin Cat T100A B Takaka Recombinant Human Fibronectin Fragment v201303 NOTE This product is for research use only It is not intended for use in therapeutic or diagnostic procedures for humans or animals Also do not use this product as food cosmetic or household item etc Takara products may not be resold or transferred modified for resale or transfer or used to manufacture commercial products without written approval from TAKARA BIO INC If you require
11. ca 100A B For Research Use Tahara RetroNectin Recombinant Human Fibronectin Fragment Product Manual v201303 RetroNectin Cat T100A B Takaka Recombinant Human Fibronectin Fragment v201303 Table of Contents li gt BesellDilOlb j 4 50 445 15 4 5454 1 2 e d ada ka tat alek at w war RA 3 l o l erelni sn SI a r Hec 4 R oe Dr r ee eee rg 4 IW Materials Required but not Provided i ii lc anne u l l l 4 U SAR Ki rol col olo Pe r stan int AANE 5 1 Preparation of RetroNectin Coated Plates w ccsssecsssecssseccssseecssseessseesees 5 2 Gene 1 TanSsd U llOa srs ara a ERRA 5 A RetroNectin bound Virus RBV Infection Method 6 B Supernatant SN Infection Method cccecsssessssssccssessssecsssssesssseecsseeessses 8 W Dn eee em DD r Dr rr nr 9 Wil Rete Product Ko A e K e K e K K K S e K n da k 9 TAKARA BIO INC URL http www takara bio com RetroNectin Cat T100A B Takaka Recombinant Human Fibronectin Fragment v201303 l Description RetroNectin reagent is a recombinant human fibronectin fragment rFN CH 296 composed of three functional domains the cell binding domain C domain heparin binding domain H domain and CS 1 sequence The fragment enhances retroviral mediated gene transduction by aiding the co localization of target cells and virions Specifically virus particles bind RetroNectin reagent via interaction with t
12. he H domain and target cells bind mainly through the interaction of cell surface integrin receptor VLA 5 and VLA 4 with the fibronectin C domain and CS 1 site respectively Through facilitating close proximity RetroNectin reagent can enhance retroviral mediated gene transfer to target cells expressing integrin receptors VLA 4 and or VLA 5 1 There are two RetroNectin mediated infection protocols the supernatant SN infection method and the RetroNectin bound virus RBV infection method 5 2 With the SN infection method cells are mixed with virus supernatant and loaded on a RetroNectin coated plate In the RBV method the retrovirus is first bound to the RetroNectin coated plate and cells are added after removing the retrovirus supernatant Removal of the supernatant reduces inhibitory molecules e g molecules secreted from the producer cells such as proteoglycans and or viral envelope proteins that can reduce the efficiency of viral mediated gene transduction 1 RetroNectin can also enhance lentiviral mediated gene transfer 2 Both methods can be used for efficient gene transduction Although the RBV infection method is widely applicable some modification might be required depending on the target cells vectors and or target genes VLA 5 a 5 1 VLA 4 a4 8 Cell binding domain C Domain RGDS Heparin binding 2 Bu TN domain H Domain ra
13. lls can be also used for resuspension URL http www takara bio com TAKARA BIO INC RetroNectin Cat T100A B Takaka Recombinant Human Fibronectin Fragment v201303 B Supernatant SN Infection Method When the virus stock solution is used the RBV method described in A is recommended but if a 4 fold dilution or more is used either the RBV or SN method may be used as equivalent gene transduction efficiency will be obtained The time required for virus infection is much shorter with the SN method than with the RBV method 1 Suspend the target cells in virus solution that has been diluted with growth medium to prepare the cell suspension 2 Add the cell suspension to the RetroNectin coated plate at a density of 0 5 2 5 x 104 cells cm2 Although the optimal cell density depends on cell size and growth rate the initial cell density should allow the cells to be actively growing or nearly confluent when gene expression is analyzed 2 3 days after transduction When infecting more cells you may increase the cell density but the cells will need to be subcultured after gene transduction Note To promote contact between the target cells and virus vectors the plate can be centrifuged after adding the cells 3 Incubate in a 37 C 5 CO2 incubator for 2 3 days 4 Collect both non adherent and adherent cells 1 Transfer the supernatant to a centrifuge tube 2 Recover remaining non adherent cells by washing the plate with PBS
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