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Transfection - Basics, Principle, Mechanism and Optimization of Mammalian Cell Transfection. Lentiviral vectors are a powerful gene delivery tool; high transfection efficiency, simple and safe to...

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A lentiviral construct containing the gene of interest along with lentiviral packaging mix is cotransfected into 293T or 293FT cells using Lipofectamine 3000 reagent. Following incubation of cells, supernatant containing lentivirus is harvested and cellular debris is removed by centrifugation.

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Lentiviruses were produced by reverse transfection of suspended 293T cells using 20 ug Polyjet and Lipofectamine 2000 are good reagents for transfection of 293T cells to produce lentivirus.

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Retrovirus Transfection Protocol. Date Submitted Submitted by. Adapted from Contributor(s). This protocol was developed to consistently produce a high titer retrovirus.

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Method. Day 1. Add 1.6 x10 4 cells in fresh medium to the number of wells needed for each construct in a 96-well plate. Duplicate or triplicate wells for each lentiviral construct and control to be used. Incubate 18-20 hours at 37 °C in a humidified incubator in an atmosphere of 5-7% CO 2.

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Recombinant Lentivirus Production Protocol Lentivirus is classified as retroviridae. The pre-integration complex of the lentivirus nuclear protein has the characteristics of phagocytosis, and the viral genome translocates to the nucleus, so that it can infect and replicate in the non-mitotic cells.

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3 Transfection Workflow Section Divider. 4 Common Transfection Methods Reagent-Based Methods Lipids Calcium phosphate Cationic polymers DEAE-dextran Activated dendrimers Magnetic...

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Use this protocol to generate lentivirus. This protocol can be used to produce lentivirus from a lentiviral vector transfected into Lenti-X 293T cells using a polyethyenimine (PEI) transfection...

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Lentiviral vectors are a powerful gene delivery tool; high transfection efficiency, simple and safe to use. Transfection - Basics, Principle, Mechanism and Optimization of Mammalian Cell Transfection.

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the protocol involving the 2nd generation packaging system has been useful. We have not attempted to reduce the total amount of transfected DNA in the protocol using the 3rd generation packaging system. Plate 1×106 target cells in 10 cm dish per transfection. [Alternatively, to use less of the viral supernatant to be

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Lentiviral transduction is an efficient method for the delivery of transgenes to mammalian cells and unifies the ease of use and speed of transient transfection with the robust expression of stable cell lines.
Figure 1. Workflow diagram of the vector production protocol. Flow diagram outlining the main steps at each day of vector production, includ - ing culturing HEK 293T cells, transfection of cells with lentiviral plasmids, changing to a serum free media and finally the purification and concen-tration of the final product.
Lentivirus packaging in 293T Cells Split cells every Monday and Friday: For a T75 flask, that is 70-80% confluent, wash cells X1 with PBS being careful not to disturb cells as they detach easily.
transfection protocol (Invitrogen). The following amount of DNA was used per cell factory: 4 mg of pMLV SIN-CMV-GFP or pMND-GFP-SN, 1.5 mg of pMD-GP (gag-pol) and 1.5 mg of pCI-VSV-G (VSV-G envelope). The pMD-GP and pCI-VSV-G plasmids were kindly provided by Dr. D. Ory (University of Washington, St. Louis). Twenty-four hours
Lentivirus Transduction Protocol. Day 1: Plate continuous (immortal/transformed) cells (2.5x10^4) in 12 Transient transfection is advantageous for fast analysis of shRNA mediated effects, and may...

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Lentiviruses were produced by reverse transfection of suspended 293T cells using 20 ug Polyjet and Lipofectamine 2000 are good reagents for transfection of 293T cells to produce lentivirus.
INTRODUCTION This protocol combines the specificity of small interfering RNA (siRNA)-mediated silencing cassettes with the versatility of lentiviral vectors to stably transduce a wide range of cell types. A short hairpin RNA (shRNA) designed against a given target is cloned into a plasmid containing the pol III promoter. Here, we optimized the HEK293T cell transfection and provided a convenient and high-productive protocol for lentivirus production. The process yielded a total amount of more than 4 × 10 8 TU of lentivirus particles per 10 cm dish in 4 days. The lentiviral vectors successfully served in vitro and in vivo RNAi experiments in our lab [ 5, 11, 12 ].