If the original lentivirus stock is 109 tuml, then you will need 0. Cd19 is widely expressed on b cells, starting from the earliest precursor cells through all stages of maturation. Um171 enhances lentiviral gene transfer and recovery of. Typically, lineagenegative cells account for 2%5% of whole bone marrow nucleated. Regulatory considerations for manufacturing and testing of. Recently, the lentivirus system is widely used to integrate sirna efficiently in a wide variety of cell lines and primary cells both in vitro and in vivo. Lentiviral vectors for immune cells targeting ncbi. Therefore, we provide an optimized protocol that is easy to implement and allow transduction. Lentiviral gene therapy for the treatment of inherited liver disease. Compared to conventionally stable cell line construction, lentivirus. For each sample, plate 100,000 human dermal fibroblast cells in fibroblast medium on a gelatin coated 35mm plastic culture dish. Most gene therapy lentiviral vector lv production platforms employ hek293t cells. Lentivirus is prepared and titered as described in rubinson et al. Lentivirus transduction protocols for reference only many factors can affect transduction efficiency.
Engineering lentiviral vectors to have the capacity to transduce specific cell types is of great interest to advance the translation of lentiviral vectors towards the. Make sure to use the polybrenecontaining media to make the cell solution in this step. Systemic delivery of lentiviral vector lv in immunocompetent mice leads to. Lentiviral transduction efficacy in n9 and bv2 cells. Lentiviral transduction of immune cells springer nature experiments.
Reconstructing the immune system with lentiviral vectors ncbi. Centrifuge at 3000 x g for 20 min and then filter the supernatant through a 0. When transducing a lentiviral construct into a cell line for the first time, a range of volume or moi should be tested. Hiv1 derived lentiviral vectors lvs transduce dcs at high efficiency but their effects on dc functions have not been carefully studied. For fresh adult cells, passage 34 is best and reprogramming efficiency declines with each passage. Furthermore, terminally differentiated cells often have a short turnover time and are. Mouse lineagenegative cells are isolated from the bone marrow of cas9 transgenic mice 3, or other strains of mice, using a lineage depletion kit according to the manufacturers instructions. Transduction efficiency varies in different cell lines, and polybrene may be toxic to some cell lines. Low passage, actively growing cells work best for infection. T cells and further cell expansion are challenging. The higher rates of transduction of clonogenic cells by lvv suggest that these vectors are more ef. While wt vsvg lentiviral particles could infect 24.
Protocol 3 lentivirus transduction into target cell. Replace the transfection medium with fresh hek medium and return to the incubator. In human ec, startling differences were observed between the three viral systems. Some additives such as polybrene can enhance the transduction efficiency. Efficient gene transduction of neurons by lentivirus with.
This protocol was adapted from lentivirus transduction of hematopoietic cells, chapter 7, in gene transfer. Wash cells with optimem and then add the peidna mix. For example, if you intend to infect 50 thousands cells at moi of 10, you need 10x50,000 5. Lentivirus particles are produced from 293t cells through transient transfection of plasmids that encode for the components of the virion. Pdf lentiviral vector gene transfer into human t cells.
Simplified production and concentration of lentiviral. In this overview we distinguish between two types of lentiviral vector targeting strategies. Anybody ever used lentivirus for transduction of cells. Persistence of gfptg apc in secondary lymphoid organs 7. Viruses can be a useful tool for this delivery, particularly in difficult to transfect cell types. Robust and persisted transduction efficiency of blood cells with lvs. Lentiviral vectors lvs developed in the past two decades for research and preclinical purposes have entered clinical trials with remarkable safety and efficacy performances.
Here we provide an overview of innovative approaches to target lentiviral vectors to cells of the immune system. Not all viral particles floating in culture medium can eventually transduce or infect the cells. Ex vivo lentiviral gene transfer can result in efficient transduction of progenitor cells 80% that can then. Antonia follenzi, laura santambrogio and andrea annoni affiliation. Publication processes organization and format errata, author corrections, retractions. Dendritic cells dcs are professional antigenpresenting cells that play important roles during human immunodeficiency virus type 1 hiv1 infection. The experiments described here demonstrate that lentivirally transduced cells can be identified by the immune system only if an immune response is raised against the transgene. Vector copy numbers vcn for rv or lv vectors for rv or lv. We show that these dc are activated by lentiviral transduction in cell cultures and in vivo. Alteration of t cell immunity by lentiviral transduction. An alternative approach is the transduction of differentiated cells.
Adenoassociated virus aav is a useful gene transfer vector because of its ability to mediate efficient gene transduction in numerous dividing and quiescent cell types, without. Robust and persisted transduction efficiency of blood cells with lvs, resulted into a broad range of target cells for immune therapeutic. Lentiviral vector production titer is not limited in hek293t by induced intracellular innate immunity. A survey of ex vivoin vitro transduction efficiency of. Anticancer immunotherapy is based on enhancing the immune response to tumor cells. Asymmetry in the assembly of the rnai enzyme complex. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Protocol 8 lentiviral transduction of hematopoietic stem. Development and clinical testing of lvs for improvement of human immunity showed major advantages in comparison to other viral vector systems. Do you have any suggestions to imprvoe the infection of the cells.
Hiv1 lentiviral vector immunogenicity is mediated by tolllike. However, some differentiated cell types, such as macrophages, are difficult to transduce 6. Good practice on the assessment of gmorelated aspects in the. Direct lentivirus injection for fast and efficient gene. Based on the efficiency of transduction of vascular cells by lentiviral vectors, we directly compared lentivirus with ad and aav2 gene delivery systems using equal mois of each virus, all of which expressed egfp from the cmv promoter. Vectofusin1, a new viral entry enhancer, strongly promotes. Since, especially the naive t cells will provide a longlasting immune reconstitution to patients these are the cells that need to be transduced for. When designing lentiviral transduction experiments, it is recommend to use a reporter vector such a lentiviral vector expressing egfp to determine optimal transduction conditions. Protocol for lentiviral transduction of human cells. Efficient delivery and sustained expression of a therapeutic.
Contains 3 lentivirus structural genes gag, pol and env, and only 1 lentivirus regulatory gene rev tat, a viral gene essential for viral selfreplication, is eliminated and transacti vation of the vector transcription is achieved by placing a strong constitutive promoter upstream of the vector transcript. We describe a method for generating lentivirus that is simple to perform and does not require the purchase of nonstandard equipment to transduce primary human t cells. Lentivirusmediated in vitro gene transfer into nile. Lentivirus mediated in vitro gene transfer into nile tilapia spermatogonial stem cells s. Cold spring harbor laboratory press, cold spring harbor, ny, usa, 2007. Direct lentivirus injection for fast and efficient gene transfer into brown and beige adipose tissue.
Besides, engineered lentiviral transfer vector that embedded a matrixattachment region mars sequence may provide positionindependent transgene expressions. Albert einstein college of medicine, 0 morris park avenue, bronx, 10461 ny, usa. Gene therapy, hiv1 derived vectors, innate immunity, adaptive immune responses, transgene abstract. Modification of dcs using lvs may lead to important applications in transplantation, treatment of. Protocol for transduction of human embryonic stem cells. If dna or rna is introduced into cells by using viral vector carriers, then the technique is called transduction, and the resulting cells are said to be transduced. This suggests that the strength of tcr signalling may be important for increased transduction efficiency.
It includes virusbased vectors such as lentiviral vectors, adeno. Crisprcas9 can be employed to rapidly engineer immune cells and oncolytic viruses for cancer immunotherapeutic applications. Control of vector transduction a critical step for controlling the product potency transduction efficiency determine upper and lower limits in early phases and refine the acceptance criteria towards late phase studies. Gently mix lentivirus, and add with 1ml pipette tip appropriate amount of virus to each well. Immune responses to lentiviral vectors bentham science. Lentiviral transduction of endothelial cells mellorlab. When i infect 293t cells with the viral particles to calculate the viral titer i get a very high efficient gfp signal on facs. Lentivirus transduction protocols for reference only. Immune response to lentiviral bilirubin udpglucuronosyltransferase gene transfer in. Thirdgeneration lentivirus vectors efficiently transduce. Lentivirus biosafety information and handling guidelines. Lentivirusmediated gene transfer in hematopoietic stem. However, antitransgene immune response results in clearance of transduced.
Start this protocol by preparing the dna with which one will later transfect ones cells to make virus. Lentiviral transduction of immune cells springer for. Efficient gene transduction of neurons by lentivirus with enhanced neuronspecific promoters. Lentivirus delivered stable gene silencing by rnai in primary cells. The ability to deliver a gene of interest into a specific cell type is an essential aspect of biomedical research. Lentivirusmediated gene transfer to human epidermis. Lentivirus transduction of spleen cells microbiology. Perform a reverse transduction by seeding 50,000 cells into each well of the 6well dish. With some cell lines, a higher moi might be needed. Next day cells may be transplanted by injection or cultured further. These cells will be added to the wells that already contain 0. Isolation and lentivirus transduction of lineagenegative cells note. Lentiviral vector production titer is not limited in.
Low viability cells are sensitive to transduction enhancer. Transfection of hek 293 cells to produce lentiviruses 24 hours before transfection, plate 2. The immune response to lentiviraldelivered transgene is modulated. Differences between transfection and transduction blog. Determine the minimal concentration to kill all nontransduced cells. Transduction means gene transfer using viral vector carriers. Lentiviral transduction unit tu to be used moi x number of cells to be infected. Es cells are cultured according to standard procedures. Ex vivo lentiviral gene transfer can result in efficient transduction of progenitor cells 80% that can then be further differentiated into immune lineage cells including t, b, dendritic, or natural killer cells. Overcoming the challenge of transduction of human tcells. Amelioration of emphysema in mice through lentiviral. Lentivirus transduction of spleen cells may202009 hello all, i am transfecting 293t cells with packaging lentiviral plasmids to generate a lentiviral particles carrying gfp and another gene.
Lentiviral crisprcas9mediated genome editing for the. Lentiviral vectors for gene transfection in car tcell. Reconstructing the immune system with lentiviral vectors. Adipocytespecific expression can be achieved with specific promoters to control expression of the target gene. Infection protocol for recombinant lentivirus 9601 medical. Capable of transduction of nondividing cells can generally transduce hard totransfect cell lines transduction efficiency can reach 100% control of delivery straightforward to deliver a single copy to cells easy to monitor copy number c. Enhanced lentiviral transduction of hsc in the presence of the vectofusin1 peptide.
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