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

Transcription

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

2 I. Description : RetroNectin Dish is a precoated culture dish (35 mmφ) with RetroNectin (Cat.#T100A/B) RetroNectin, 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, RetroNectin 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 RetroNectin coated plate, while in the improved method (RetroNectin -bound virus infection method), the retrovirus is first bound to the RetroNectin 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 RetroNectin Dish (35 mmφ Dish) 10 dishes Note: The dish existing crystalized salt can be used without any probrems. II-2. Storage 4 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 RetroNectin (CH-296) Fibronectin SH RGDS CS-1 SH NH COOH SS Fibrin Collagen DNA Cell Heparin Blue: binding domain Heparin Cell (III CS) 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 RetroNectin. In this way the localized concentrations of both are increased, which as a result is thought to enhance gene transduction. 3

4 III. Reagents and Instruments Required but Not Supplied in the Kit: [Equipment] 1. Electric pipetter 2. Pipetter 3. Sterilized pipette 4. Sterilized chip with filter 5. Safety cabinet or clean work station 6. Microscope 7. CO2 incubator [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 IV. Gene Transduction: There are two methods of gene transduction using RetroNectin : RetroNectin -bound virus (RBV) infection method and supernatant (SN) infection method. In the RBV infection method, a retrovirus is first bound to the RetroNectin coated plated, 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 RetroNectin 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 RetroNectin which has virus binding activity. Note : An example of retrovirus vector preparation: Using transient production system of Retrovirus Packaging Kit Eco/Ampho (Cat.#6160/6161) and pdon-ai-2 DNA/pDON-AI-2 Neo DNA (Cat.#3654/3653), you can quickly and easily prepare a retrovirus vector within one week. 4

5 V. Transduction Protocol : A. RetroNectin -bound Virus (RBV) Infection Method A-1. Preparation of virus binding plate 1. Pre-load retrovirus supematant carrying your objective gene onto a RetroNectin Dish at 125 ~ 250 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 RetroNectin. During this incubation retrovirus vector will bind to the H-domain of RetroNectin. 3. Discard the virus supernatant, taking care that the virus bound on the RetroNectin 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. A-2. Virus Infection Prepare the target cells while the retrovirus vectors are binding to the RetroNectin Dish. 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 0.2 ~ cells/ml. 2. Remove the solvent from the virus bound plates prepared by A-1. After removal of the solvent do not keep it stand for long time. Immediatly add target cells in growth medium at the concentration of 0.5 ~ 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 ~ 3days 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. 3. Incubate at 37 under 5 % CO2 for 2 ~ 3 days. 4. Collect both non-adherent and adherent cells by the following steps. (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 pipetteing 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. 5

6 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 RetroNectin Dish at the concentration of 0.5 ~ 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. 3. Incubate at 37 under 5 % CO2 for 2 ~ 3 days. VI. 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, VII. Related Products : Recombinant retroviral vecto r: pdon-ai DNA (Cat.#3650) Retrociral vector : Retrovirus Packaging Kit Ampho (Cat.#6161) Retrovirus Packaging Kit Eco (Cat.#6160) Other : RetroNectin (Cat.#T100A/B) 6

7 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. [M62] RetroNectin This product is covered by the claims of U.S. Patent No. 5,198,423 and its foreign counterpart patent claims. NOTE: This product is intended to be used for research purpose only. They are not to be used for drug or diagnostic purposes, nor are they intended for human use. They shall not to be used products as food, cosmetics, or utensils, etc. Takara products may not be resold or transfered, 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 call at or contact from our website at 7 Phone: Fax: