Lentiviral transduction of immune cells springerlink. Lentiviral vectors lvs are replicationdefective hybrid enveloped viral particles made by a minimal set of capsid proteins of the parental virus in most cases hiv, a surface protein of an unrelated virus referred to as pseudotype and a recombinant viral genome, comprising the cis acting elements of the parental virus strictly required for gene transfer purposes, and a. When designing lentiviral transduction experiments, it is recommend to use a reporter vector such a lentiviral vector expressing egfp to determine optimal transduction conditions. Lentivirus biosafety information and handling guidelines. Protocol 3 lentivirus transduction into target cell lines. Lowenstein1,4 abstract lentiviral vectors are promising tools for gene therapy in the cns. Second line of defense fight local infection with inflammation process. Prior to transduction, establish the puromycin kill curve. Protocol 8 lentiviral transduction of hematopoietic stem cells. Protocol for transduction of human embryonic stem cells.
Previously, retrovirus vectors could not be used for these cell types. Transfection is efficient on adherent immortalized cells but primary and stem cells require transduction. If you plan to do facs analysis posttransduction, and if possible, include as control a similar vector without gfp, and nontransduced cells as well all from the same original batch of cells, in. To obtain the desired infection rate low and high moi. Lentiviral transduction of endothelial cells mellorlab. A number of different modifications in vector design have increased biosafety and transgene expression. Rgr expression in arpe19 cells increased steadily for up to 10 days after transduction and was stable for at least 6 months.
With some cell lines, a higher moi might be needed. Upon disseminated adenovirus infection, the immune system senses. Cold spring harbor laboratory press, cold spring harbor, ny, usa, 2007. Since, especially the naive t cells will provide a longlasting immune reconstitution to patients these are the cells that need to be transduced for effective gene therapy. Replace the transfection medium with fresh hek medium and return to the incubator. Our work over the past eight years has focused on the use of hiv1 lentiviral vectors lentivectors for the genetic modification of dendritic cells dcs to control their functions in immune modulation. What links here related changes upload file special pages permanent link. Spinoculation of suspension cells system biosciences. Taichman department of oral biology and pathology, stony brook university, stony brook, new york for longterm cutaneous gene therapy, the therapeutic gene must be targeted to stem cells and be stably trans. Lentiviral transduction of hspc and infusion of genecorrected cells into patients pretreated with reduced intensity conditioning. A lot of work has been done to demonstrate the efficacy of gene therapy in animal models that mimic situations in humans. The innate immune system is one of the two main immunity strategies found in vertebrates the. On the day of transduction, thaw your lentiviral stock and dilute the lentiviral stock into complete. 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.
Infection protocol for recombinant lentivirus 9601 medical. Modification of dcs using lvs may lead to important applications in transplantation, treatment of cancer. However, efficient transduction of therapeutic gene into human t cells and further cell expansion are challenging. Robust and persisted transduction efficiency of blood cells with lvs, resulted into a broad range of target cells for immune therapeutic. Lentiviral transduction of immune cells request pdf. Common themes in microbial pathogenicity revisited pdf. Make sure to use the polybrenecontaining media to make the cell solution in this step. Lentivirus transduction protocols for reference only. When cell membranes from the arpe19 cells were incubated with 3halltransretinal, the chro. Natural killer nk cells are the major lymphocytic population of the innate immune system1. Nk cells also play a central role in the development of tolerance through the secretion of potent cytokines and chemokines5. Therefore, we provide an optimized protocol that is easy to implement and allow transduction with high efficiency and reproducibility.
The transduced arpe19 cells produced 100fold higher amounts of rgr protein than the transduced cos7 cells. Production and concentration of lentivirus for transduction. The transduction volume is established according to several parameters. Simply add premade lentiviral particle into the diluted cells at a ratio of. Dextran enhances the lentiviral transduction efficiency of. Lentiviral transduction efficacy in n9 and bv2 cells. This protocol was adapted from lentivirus transduction of hematopoietic cells, chapter 7, in gene transfer. These cells will be added to the wells that already contain 0. Lentivirus mediated gene transfer to human epidermis soosan ghazizadeh,1 anne b. Alteration of t cell immunity by lentiviral transduction of.
Due to their potent ability to target and eliminate tumor. Blood contains red blood cells, for carrying oxygen to tissues and organs, and white or immune cells, for. Expression of a recombinant human rgr opsin in lentivirus. Immune modulation by genetic modification of dendritic cells. Dendritic cells dcs are the sentinels of the immune system, and thus specialized in transporting foreign antigen to t cells and initiating activation of innate and adaptive immune responses.
Nk cells function as the firstline defenders of the host immune response against tumors and infections2,3,4. Plate cells so that cell density will be 2550% confluent at the time of transduction. Nov 12, 20 lentiviral vectors have emerged as efficient vehicles for transgene delivery in both dividing and nondividing cells. Lentiviral vectors lvs, such as those derived from the human immunodeficiency virus hiv are capable of infecting nondividing cells through mitosisindependent transport of the viral dna into the nucleus. This protocol may be used with 96well, 48 well, 24well, 12well and 6well plates. In this work, we first explore how dcs sense viral pathogens and stimulate antigenspecific t cell responses. If you plan to do facs analysis post transduction, and if possible, include as control a similar vector without gfp, and nontransduced cells as well all from the same original batch of cells, in. Lentivirusbased gene therapy of hematopoietic stem cells. Mathematical modeling of the immune system in homeostasis. Now it is clear that use of the survival cytokines, il2 or il7, allows an efficient lentiviral vector gene transfer and could preserve a functional tcell repertoire. Lentivirus transduction protocols for reference only many factors can affect transduction efficiency. Phagocytes, immune cells and the soluble components of the immune system cannot gain access to pockets of fluid or poorly vascularised tissue.
The final concentration of puromycin should be from 110. Three children with was, who had been shown by genotyping to carry severe mutations in the xlinked was gene and who did not have compatible allogeneic donors, were enrolled in the phase iii clinical trial table 1. One day prior to the transduction, seed the cells of interest into seven wells e. 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. Dendritic cells dcs are professional antigenpresenting cells that play important roles during human immunodeficiency virus type 1 hiv1 infection. The cells should be about 80% confluence at the time of transfection. Pathogenic lentiviruses cause severe immunologic or neurologic diseases in humans and other mammals.
Simplified illustration of the lentiviral transduction mechanism. Differences between transfection and transduction blog. Transduction efficiency should be checked 72 hrs following. 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. These data support the further development of lentiviral vectors to transduce dcs with genes encoding ags or immunomodulatory adjuvants to generate and control systemic immune responses. Stability of lentiviral vectormediated transgene expression in the brain in the presence of systemic antivector immune responses evelyn abordoadesida,1,2 antonia follenzi,3 carlos barcia,1 sandra sciascia,1 maria g.
Modulation of immune responses in lentiviral vectormediated. However, despite these advances, the transduction of primary human t cells is still challenging and methods to achieve efficient gene transfer are often expensive and time. Examine cells each day and change to fresh puromycincontaining media every other day. Some additives such as polybrene can enhance the transduction efficiency. The innate immune system in dendritic celltargeted. May, 2015 wash cells with optimem and then add the peidna mix.
Immune responses to lentiviral vectors bentham science. Production of lentiviral vectors for transducing cells from. Cells were infected with gfp lentivirus for 48 hours in the presence of polybrene or the viraductin lentivirus transduction kit. However, this protocol is not able to extract cells during infection. Lentiviral vectors for immune cells targeting ncbi. Stability of lentiviral vectormediated transgene expression. Use a lentiviral vector to transduce target cells which naturally integrates its dna into the host cell genome in a random manner. The infection protocol for suspension cell is recommended for transduction of suspension cells and includes an additional spin step which concentrates the virus at the target cell surface to increase transduction efficiency. Lentiviral transduction of immune cells springer for. Overcoming the challenge of transduction of human tcells. Mathematical modeling of the immune system in homeostasis, infection and disease. Spinoculation of suspension cells this protocol is for transduction of suspension cells jurkat t cells, pbmc, pbl, b cells etc.
The immune system uses several tools to fight infection. Seed 293t cells at 3040% confluence to 10 100mm tissue culture dishes 3 x 10 6 cells dish in culture medium. Viral transduction of mammalian cells entered by kevin janes janes lab protocols 5111 1 when referring to this protocol, please cite. As per the paper, naive t cells are required to be prestimulated with il7 for 612 days for efficient lentiviral transduction, whereas. Protocol for lentiviral transduction of human cells. Simplified production and concentration of lentiviral vectors. Lentiviral transduction of b cells may202009 hello all, i am transfecting 293t cells with packaging lentiviral plasmids to generate a lentiviral particles carrying gfp and another gene. Centrifuge at 3000 x g for 20 min and then filter the supernatant through a 0. Immunization with lentiviral vectortransduced dendritic. As a gene delivery system, lentivirus derived vectors can efficienctly deliver transgenes into a host cell genome and infect both dividing and nondividing cells 2, making them powerful molecular biology research tools. Not all viral particles floating in culture medium can eventually transduce or infect the cells. Reconstructing the immune system with lentiviral vectors ncbi. Label plates from 110 and add appropriate puromycincontaining media to cells. On the other hand, transfection or transduction of rna is always transient.
The day before transduction, trypsinize and count the cells, plating 0. Viraductin lentivirus transduction kit cell biolabs, inc. A major challenge has concerned development of gene transfer systems capable of efficient cell transduction and transgene expression without harm to the recipient. When transducing a lentiviral construct into a cell line for the first time, a range of volume or moi should be tested.
Il7 based protocol is better for cd4 t cell transduction. Discard the other half of cells or keep to passage if more platescolonies are needed. The ability to fight infection, illness, and disease. Recruiting immune cells to sites of infection through the production of chemical. Dcs are key professional antigen presenting cells which regulate the activity of most effector immune cells, including t, b and nk cells. These results suggest that compared with agpulsed dcs, vaccination with lentiviral vectortransduced dcs may achieve more potent antitumor immunity. The standard digestion procedure to isolate intestinal immune cells. Alternatively, differentiated immune cells can themselves be transduced ex vivo with lentiviral vectors. 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. Primary t cell lentiviral transduction researchgate. Determine the minimal concentration to kill all nontransduced cells.
604 50 621 488 1370 1336 924 301 1447 231 668 1434 1185 419 593 1475 592 404 522 1019 934 1313 1403 480 62 366 183 10 1223 1144 29 1369 1062 730 632 135 1473 823 1314 1262 401 825 205 763 58 379 795 757 364