Table of Contents. II. Components and Storage...2. III. Reagents and Instruments Required but not Supplied in the Kit...3

Transcription

1 Table of Contents I. Description...2 II. Components and Storage...2 III. Reagents and Instruments Required but not Supplied in the Kit...3 IV. Preparation of Coated Plates...4 V. Gene Transduction...4 VI. Transduction Protocol...5 A. -bound Virus (RBV) Infection Method...5 A-1. Preparation of virus binding plate by standing...5 A-2. Preparation of virus binding plate by centrifugation...5 A-3. Virus Infection...6 B. Supernatant (SN) Infection Method...7 VII. References...8 VIII. Related Products...8 1

2 I. Description :, a recombinant human fibronectin fragment CH-296, is composed of three functional domains, i.e. cell-binding (C-domain), heparin-binding (H-domain), and CS-1 sequence. It enhances retroviral mediated gene transduction by co-localizing target cells and virions on the rfn-ch296 molecules. The C-domain and CS-1 sequence interact with target cells through integrin receptors VLA-5 and VLA-4, respectively, and virions can be localized upon H-domain composed of three type III repeats (III12-III13-III14). So, will be useful for retrovirus mediated gene transfer to the target cells expressing integrin receptors VLA-4 and/or VLA-5. Recently we reported a method to remove inhibitory molecules that have been found in the retrovirus supernatant. These molecules secreted from the producer cells such as proteoglycans and/or retroviral envelope proteins are undesirable for retroviral mediated gene transduction. The existence of such substances may reduce retroviral gene transfer efficiency. Therefore it is important to remove such inhibitors from infection environment. In the previous method (supernatant infection method) cells are mixed with virus supernatant and loaded on a coated plate, while in the improved method (-bound virus infection method), the retrovirus is first bound to the coated plate, and cells are added to conduct infection after removing the retrovirus supernatant that contains inhibitory molecules. * The previous method can also be used for efficient gene transduction. Although the improved method is widely applicable, some modification might be required depending on the target cells, vectors and objectives, etc. II. Components and Storage : II-1. Component 0.5 mg (0.5 ml) (Cat. #T100A) 2.5 mg (2.5 ml) (Cat. #T100B) Each product is a sterilized solution (1 mg/ml) II-2. Storage -20 Caution : Freeze-thaw cycle can be repeated within 10 times. Don't mix the solutions vigorously (Do not use Vortex mixer). 2

3 Target Cell VLA-5(α 5 β 1 ) receptor VLA-4(α 4 β 1 ) Cell-binding domain (C-Domain ; RGDS) Heparin-binding domain(h-domain) CS-1 (CH-296) RGDS CS Fibronectin SH Cell RGDS Heparin CS-1 SH NH Fibrin Collagen DNA Cell Heparin Blue: binding domain Heparin Cell (III CS) COOH S S Fibrin The cell binds to the CS-1 site, a VLA-4 ligand, and to the cell attachment domain, a VLA-5 ligand, while the virus vector binds to the heparin binding domain to colocate on. In this way the localized concentrations of both are increased, which as a result is thought to enhance gene transduction. III. Reagents and Instruments Required but Not Supplied in the Kit : [Equipment] 1. Non-treated tissue culture plates or dishes [e.g. BD Multiwell cell culture plate 24-well (BD code ), 6-well (BD code )] 2. Electric pipetter 3. Pipetter 4. Sterilized pipette 5. Sterilized tips with filter 6. Safety cabinet or clean workstation 7. Microscope 8. CO2 incubator 9. Centrifuge (adaptable to 96 well plate) [Reagents] 1. Sterilized PBS ( - ) (Phosphate Buffered Saline) 2. HBSS/Hepes (Hank's Balanced Salt Solution supplemented with 2.5% (v/v) 1M Hepes) 3. 2% BSA (BSA Fraction V)/PBS Solution 3

4 IV. Preparation of Coated Plates : 1. Prior to coating, adjust Solution to a desired concentration (ranging 20 to 100 μg protein/ml * ) by diluting with sterilized phosphate buffered saline (PBS). * : A hint on calculating coating concentration : When 2.25 ml of solution at the concentration of 20 μg protein/ml is placed into 35 mm diameter well (9 cm 2 ), the well is layered with 5 μg protein of per square centimeter (5 μg/cm 2 ). Note : solution should not be filtrated after dilution with PBS and also care should be taken when filters other than low protein binding type are used. tends to be adsorbed by filtration. 2. Dispense an appropriate volume * of sterile solution into each plate and allow the covered plate to stand for 2 hours at room temperature in a clean bench or 4 for overnight. * : It is recommended to dispense 0.5 ml into each well of a 24-well plate or 2 ml into each well of a 6-well plate respectively. Note : Non-treated tissue culture plates or dishes should be used in this step. (The quality of those should be cell culture grade.) 3. Remove solution and then add an appropriate volume * of sterile PBS containing 2% bovine serum albumin (BSA, Fraction V) into each well for blocking. Allow the plate to stand at room temperature for 30 minutes. * : It is recommended to add 0.5 ml for each well of a 24-well plate or 2 ml for each well of a 6-well plate respectively. 4. Remove the BSA solution, and wash wells once with an appropriate volume * of Hank's Balanced Salt Solution supplemented with 2.5% (v/v) 1M Hepes (HBSS/Hepes) or PBS. Now the plate has been coated with and is ready for use. The coated plate can be stored at 4 for one week if the plate is sealed with Parafilm. 4

5 V. Gene Transduction : There are two methods of gene transduction using : -bound virus (RBV) infection method and supernatant (SN) infection method. In the RBV infection method, a retrovirus is first bound to the coated plate, and the target cells are added after removing the virus supernatant. In the SN Method, the virus solution and target cells are added to the coated plate after mixed. If the virus is transiently prepared with a retrovirus vector, gag-pol and env gene expression vectors using 293T or other cells *, or if the virus is recovered from producer cells, the collected virus solution is the cell culture supernatant. For this reason the virus solution contains contaminating substances of the culture supernatant. If this virus solution is used for infection, the expected gene transduction efficiency might not be obtained due to infection inhibitory molecules contained in the contaminants. In such case the RBV method is recommended. In this method the infection inhibitory molecules can be removed because the viruses are purified by binding viruses to which has virus binding activity. VI. Transduction Protocol : *Note : An example of retrovirus vector preparation : Using transient production system of Retrovirus Packaging Kit Eco/Ampho (Cat. #6160/6161) and pdon-ai-2 vector (Cat. #3654/3653), you can quickly and easily prepare a retrovirus vector within one week. A. -bound Virus (RBV) Infection Method A-1. Preparation of virus binding plate by standing 1. Pre-load retrovirus supernatant carrying your objective gene onto a coated plate or dish at μl/cm Incubate for 4 to 6 hours at 32 or 37 in a 5% CO2 incubator to promote attachment of the virus particles onto the. During this incubation retrovirus vector will bind to the H-domain of. 3. Discard the virus supernatant, taking care that the virus bound on the should not dry, and wash the plate or dish with an appropriate volume of PBS or PBS containing 0.1-2% albumin (BSA or HSA). By this step the undesirabe substances existing in the retrovirus supernatant can be removed.then perform virus infection to the cells according to A-3. A-2. Preparation of virus binding plate by centrifugation If the titer of the retrovirus vector is high enough, binding of the viruses onto the can be promoted by standing still as described in A-1. However, if the titer of the retrovirus is low, or if you require highly efficient gene transduction to the cells, the following method of binding the virus onto the by centrifugation is also effective. In this case the time required to bind the retrovirus can be shortened to 2 hours from the 4 to 6 hours by standing method. A plate or tube that can tolerate centrifugation for 2 hours at 32, 1,000-2,000 g is required in this method. For example, a non-treatment multi-well plate, polystyrene centrifuge tube or a polypropylene centrifuge tube can be used. But please note there is a possibility of aerosol formation. 5

6 1. Add the retrovirus stock solution * or diluted solution carrying your objective gene to the coated plate or tube at μl/cm 2. * : The volume that can be added to the plate or tube varies depending on its shape. For a 6-well plate the upper limit should be set at 3 ml (320 μl/cm 2 ). It is different from the case of standing method, when the retrovirus are bound to the by centrifugation using the retrovirus stock solution, some infection inhibitory molecules cannot be removed by washing with PBS. For this reason the efficiency of gene transduction to the cells might be reduced. In such case it is recommended that the virus stock solution be used after dilution with growth medium. However, optimization of condition is required to decide the suitable dilution rate. 2. Set the plate or tube to which the retrovirus was added in 1. into a centrifuge pre-warmed to 32 and centrifuge for 2 hours at 32, 1,000-2,000 g to facilitate attachment of virus particles onto the. During this incubation and centrifugation the retrovirus vector binds to. 3. Discard the virus supernatant, taking care that the virus bound on the should not dry, and wash the plate or dish with an appropriate volume of solvent such as PBS or PBS containing 0.1-2% albumin (BSA or HSA). Then perform virus infection to the cells according to A-3. A-3. Virus Infection Prepare the target cells while the retrovirus vectors are binding to the plate. It is important that target cells should be in log phase growth and expressing integrin receptors VLA-4 and/or VLA-5. If your target cells are hematopoietic stem cells, pre-stimulation by cytokine cocktails might be necessary. The composition of cytokine cocktails will be determined to fit in your specific research protocols. Examples are cited in reference 3 and Collect the target cells and count the number of living cells. Then suspend the cells in the growth medium at a concentration of cells/ml. 2. Remove the solvent from the virus bound plates prepared by A-1 or A-2. After removal of the solvent do not keep it stand for long time. Immediately add target cells in growth medium at the concentration of cells/cm 2. Although the optimal cell density depends on target cell size or growth rate, it is recommended that the initial cell density should be determined in such a way as cells should be in the state of briskly growing or nearly confluent when you analyze the transduced gene expression 2-3 days after the transduction. If you wish to transfer genes to more cells, cell density can be increased but the cells will need to be subcultured after gene transduction. Note : In order to promote contact between the target cells and virus vectors, the plate (plate only) can be centrifuged after adding the cells. 3. Incubate at 37 under 5% CO2 for 2-3 days. 4. Collect both non-adherent and adherent cells by the following steps. 6

7 (1) Transfer the above 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 (CDB ; enzyme free, PBS based) or trypsin-edta solution, following manufacture's instructions. (4) Combine cells obtained from steps described above (1) to (3) in the same centrifuge tube, and centrifuge to recover cell fraction. (5) Rinse the cells with HBSS/Hepes twice by centrifugation, and suspend the cells in HBSS/Hepes for further use. Note : In case of several cell lines, the adherent cells may be also collected by pipetting only. At the step (5), the buffer or medium suitable for the customer's purpose can be also used to resuspend the cells, instead of HBSS/ Hepes. B. Supernatant (SN) Infection Method When the virus stock solution is used, the RBV method described in A is recommended, but if 4-fold dilution or more is used either the RBV or SN method may be selected as preferred since equivalent gene transduction efficiency will be obtained. The time required for virus infection is much shorter in the SN method than in 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 coated plate at the concentration of cells/cm 2. Although the optimal cell density depends on target cell size or growth rate, it is recommended that the initial cell density should be determined in such a way as cells should be in the state of briskly growing or nearly confluent when you analyze the transduced gene expression 2-3 days after the transduction. If you wish to transfer genes to more cells, cell density can be increased but the cells will need to be subcultured after gene transduction. Note : In order to promote contact between the target cells and virus vectors, the plate can be centrifuged after adding the cells. 3. Incubate at 37 under 5% CO2 for 2-3 days. 7

8 VII. References : 1. Kimizuka F, Taguchi Y, Ohdate Y, Kawase Y, Shimojo T, Hashino K, Kato I, Sekiguchi K, and Titani K. (1991) Production and characterization of functional domains of human fibronectin expressed in Escherichia coli. J. Biochem. 110, Hanenberg H, Xiao XL, Dilloo D, Hashino K, Kato I, and Williams DA. (1996) Colocalization of retrovirus and target cells on specific fibronectin fragments increases genetic transduction of mammalian cells. Nat Med. 2, 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, Pollok KE, Hanenberg H, Noblitt TW, Schroeder WL, Kato I, Emanuel D, and Williams DA. (1998) High-efficiency gene transfer into normal and adenosine deaminasedeficient T lymphocytes is mediated by transduction on recombinant fibronectin fragments. J. Virol. 72, Chono, H., Yoshioka, H., Ueno, M. and Kato, I. (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, VIII. Related Products : Recombinant retroviral vector : pdon-ai-2 Neo DNA (Cat. #3653) pdon-ai-2 DNA (Cat. #3654) Retroviral vector : Retrovirus Packaging Kit Ampho (Cat. #6161) Retrovirus Packaging Kit Eco (Cat. #6160) Other : Dish (Cat. #T110A) Retrovirus Titer Set (for Real Time PCR) (Cat. #6166) NOTICE TO PURCHASER : LIMITED LICENSE [L9] 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] Expansion Method This product is the subject of the pending U.S. patent application and its foreign counterparts. 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 licenses for other use, please contact us by phone at or from our website at 8 Phone: Fax: