Name: Lentiviral Packaging and Viral Titering for Genome-Scale shRNA Screens
Uploaded: 2023-08-25T13:30:00
Description: Watch this Scientific Journal Video about Lentiviral Packaging and Viral Titering for Genome-Scale shRNA Screens at JoVE.com

lentiviral packaging and viral titering for genome-scale shrna screens

Genome-Scale Screening to Identify and Compare Therapeutic Targets in Tumors

Malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive spindle cell neoplasms that arise in association with the tumor susceptibility syndrome neurofibromatosis type 1 (NF1), sporadically in the general population and at sites of previous radiotherapy1,2,3. NF1 patients are born with a wild-type copy of the NF1 tumor suppressor gene and a second NF1 allele with a loss-of-function mutation. This state of haploinsufficiency renders NF1 patients susceptible to a second loss-of-function mutation in their wild-type NF1 gene, which triggers tumorigenesis. When this &#34;second hit&#34; NF1 mutation occurs in a cell in the Schwann cell lineage, the resulting tumor is either a dermal neurofibroma arising in the skin or a plexiform neurofibroma that develops in large nerves or nerve plexuses. Although the pathology of dermal and plexiform neurofibromas is identical, their biologic behavior is quite different-although both dermal and plexiform neurofibromas are benign, only plexiform neurofibromas can undergo transformation and give rise to MPNSTs. In addition to the loss of neurofibromin, the Ras GTPase-activating protein encoded by the NF1 gene, MPNSTs carry mutations of multiple other tumor suppressor genes, including TP534,5,6,7, CDKN2A8,9, and PTEN10, mutations of genes encoding components of&#160;polycomb repressive complex 211,12 (PRC2; the SUZ12 and EED genes) and aberrant expression of receptor tyrosine kinases1,2. Mutations of NF1 and the other genes noted above are also present in sporadic and radiation-induced MPNSTs11,12.
While these advances in our understanding of the genomic abnormalities in MPNSTs have been invaluable for understanding their pathogenesis, they have not yet resulted in the development of effective new therapies for MPNSTs. A major barrier impeding the development of new treatments is the fact that MPNSTs are rare cancers. Because of this, it is difficult to obtain the large number of patient samples that are required for global analyses defining key driver mutations such as those undertaken by The Cancer Genome Atlas (TCGA). In our experience, accumulating even a modest number of human MPNST specimens can take years. To overcome such limitations, many investigators studying other rare cancer types have turned to the use of cross-species comparative oncogenomics to identify essential driver gene mutations, define the essential cytoplasmic signaling pathways in their tumor of interest, and identify new therapeutic targets. Since the signaling pathways that are essential for tumorigenesis are highly conserved between humans and other vertebrate species, applying functional genomics approaches such as genome-scale shRNA screens can be an effective means of identifying these new driver mutations, signaling pathways, and therapeutic targets13,14,15,16,17,18,19, particularly when studying rare human tumor types that are available in limiting numbers20.
In the methodologies presented here, we describe this approach to performing genomic profiling in human MPNST cell lines and early passage MPNST cultures derived from P0-GGF&#946;3 mice, a genetically engineered mouse model (GEM) in which Schwann cell-specific overexpression of the growth factor neuregulin-1 (NRG1) promotes the pathogenesis of plexiform neurofibromas and their subsequent progression to MPNSTs21,22,23. The first step in this approach is to identify candidate driver genes in P0-GGF&#946;3 MPNSTs, human MPNST cell lines, and surgically resected human MPNSTs. To functionally validate the signaling pathways affected by these mutations, we then use genome-scale shRNA screens to identify the genes required for proliferation and survival in human and mouse MPNST cell lines. After identifying the genes required for proliferation and survival, we then identify the druggable gene products within the collection of &#34;hits&#34; using the Drug Gene Interaction Database. We also compare the &#34;hits&#34; in human and mouse MPNST cells, to determine whether the GEM model and human MPNSTs demonstrate similar dependence on the same genes and signaling pathways. Identifying overlaps in the genes required for proliferation and survival and the affected signaling pathways serves as a means of validating the P0-GGF&#946;3 mouse model at a molecular level. This approach also emphasizes the effectiveness of combining human and mouse screens to identify novel therapeutic targets, where the mouse model can serve as a complement to the human screens. The value of this cross-species approach is particularly apparent when looking for therapeutic targets in rare tumors, where human tumors and cell lines are difficult to obtain.

Author Spotlight: Finding New Therapeutic Targets for Malignant Peripheral Nerve Sheath Tumor Through Genome-Scale shRNA Screens 

Genetic Profiling and Genome-Scale Dropout Screening to Identify Therapeutic Targets in Mouse Models of Malignant Peripheral Nerve Sheath Tumor

We are striving to understand the pathogenesis of sporadic and NF1-associated malignant peripheral nerve sheath tumors. Our goal is to logically develop new treatments for these highly aggressive neoplasms. Technologies we are currently implementing to advance our research include unbiased methods like genome-scale shRNA screens, as well as single-cell sequencing, BaseScope in situ probes, and bioinformatics.Using the various current approaches, we have identified multiple receptors that are essential for malignant peripheral nerve sheath tumors'proliferation and survival, as well as being therapeutically targetable. These receptors include the neuregulin receptor, ErbB3, and lysophosphatidic acid receptor 3. The advantage of using this approach lies in its capacity to examine the proliferative significance of over 15, 000 genes in MPNST cancer cell lines.This is an unbiased approach that identifies unexplored targets in the treatment of MPNSTs. Moving forward, our laboratory's focus will concentrate on validating the targets identified in our screen. This includes using inhibitors on cell lines and culture and ultimately, translating that to animal work.

Watch this Scientific Journal Video about Lentiviral Packaging and Viral Titering for Genome-Scale shRNA Screens at JoVE.com

Lentiviral Packaging and Viral Titering for Genome-Scale shRNA Screens  

On day zero plate 10 million 293T cells in 30 milliliters of antibiotic-free DMEM containing 10%FBS in a 15 centimeter dish. Prepare a total of 10 such dishes. On the next day, which is day one, confirm that the cells are 80%confluent and ready for transfection.Then in a 50 milliliter conical tube sequentially, add 600 microliters of 0.5 micrograms per microliter packaging plasmid mix and 60 microliters plasmid barcode library. In a separate 15 milliliter conical tube mix 900 microliters of transfection reagent and 12 milliliters DMEM by vortexing. Add 12.9 milliliters of this mixture to the DNA mix and just flick to combine.Without mixing any further, incubate the mix at room temperature for 15 minutes. Then drop wise add 2.5 milliliters of the incubated mixture to each 15 centimeter dish containing the 293T cells and incubate the cells overnight in a tissue culture incubator. On day three, harvest the virus by passing the media through a 0.2 micron filtration unit.Aliquot the filtrate containing viral particles into 15 milliliter conical tubes. Additionally, prepare five one milliliter aliquots of filtered virus in cryo vials for viral titering. To titer the lentiviral pools, add 65 microliters of Cationic polymer to a sterile glass flask containing 65 milliliters of tumor cell growth media.Pipette one milliliter of the polymer containing media per well into 11 six-well tissue culture plates. Next trypsinize the early passage tumor cells. After counting the cells using a preferred method, transfer the cells into the six-well plates.Thaw the one milliliter aliquots of the 48 hour lentivirus from the freezer in a water bath at 37 degrees Celsius. Follow the table to prepare the infection for each viral module.

lentiviral-packaging-and-viral-titering-for-genome-scale-shrna-screens

Research

JoVE Journal

Cancer Research

Malignant Peripheral Nerve Sheath Tumors (MPNSTs) are derived from Schwann cells or their precursors. In patients with the tumor susceptibility syndrome neurofibromatosis type 1 (NF1), MPNSTs are the most common malignancy and the leading cause of death. These rare and aggressive soft-tissue sarcomas offer a stark future, with 5-year disease-free survival rates of 34-60%. Treatment options for individuals with MPNSTs are disappointingly limited, with disfiguring surgery being the foremost treatment option. Many once-promising therapies such as tipifarnib, an inhibitor of Ras signaling, have failed clinically. Likewise, phase II clinical trials with erlotinib, which targets the epidermal growth factor (EFGR), and sorafenib, which targets the vascular endothelial growth factor receptor (VEGF), platelet-derived growth factor receptor (PDGF), and Raf, in combination with standard chemotherapy, have also failed to produce a response in patients. 
In recent years, functional genomic screening methods combined with genetic profiling of cancer cell lines have proven useful for identifying essential cytoplasmic signaling pathways and the development of target-specific therapies. In the case of rare tumor types, a variation of this approach known as cross-species comparative oncogenomics is increasingly being used to identify novel therapeutic targets. In cross-species comparative oncogenomics, genetic profiling and functional genomics are performed in genetically engineered mouse (GEM) models and the results are then validated in the rare human specimens and cell lines that are available. 
This paper describes how to identify candidate driver gene mutations in human and mouse MPNST cells using whole exome sequencing (WES). We then describe how to perform genome-scale shRNA screens to identify and compare critical signaling pathways in mouse and human MPNST cells and identify druggable targets in these pathways. These methodologies provide an effective approach to identifying new therapeutic targets in a variety of human cancer types.

We have developed a cross-species comparative oncogenomics approach utilizing genomic analyses and functional genomic screens to identify and compare therapeutic targets in tumors arising in genetically engineered mouse models and the corresponding human tumor type.

Malignant Peripheral Nerve Sheath Tumors (MPNSTs) are derived from Schwann cells or their precursors. In patients with the tumor susceptibility syndrome ...

Lentiviral Infection of MPNST Cells and Genomic DNA Isolation for Genome-Scale shRNA Screens

For lentiviral infection, plate the trypsinized MPNST target cells after counting them, maintaining 2.5 million cells per 15 centimeter dish. Transduce the cells with the thawed virus at a 0.5 multiplicity of infection, in the presence of five micrograms per milliliter of the cationic polymer. Trypsinize the cells three days after the addition of puromycin.Centrifuge half of the cell population at 200 G for five minutes in a tabletop centrifuge. After re-plating the other half of the cell population, grow them for approximately seven population doublings before harvesting and centrifuging as previously demonstrated. Divide the re-suspended cell pellet corresponding to the desired time into two 15 milliliter polymethylpentene tubes.Add 500 microliters of 10%SDS per tube. Mix and incubate at room temperature for five minutes. Place the tubes in a DNA shearing device to sonicate the DNA.Following the addition of phenol and chloroform. Mix well by vortexing vigorously at the maximum possible speed for 45 to 60 seconds. Transfer three milliliters of the resulting clear upper phase from each tube to another fresh 15 milliliter tube.Add 0.5 milliliters of three molar sodium acetate and four milliliters of isopropanol and mix well. After centrifuging the tubes for 30 minutes at 7, 200 G and 20 degrees Celsius and discarding the supernatant, add 0.5 milliliters of 70%ethanol to the pellet and dislodge it by pipetting up and down. Combine the re-suspended pellets from both tubes into a 1.5 milliliter centrifuge tube.Perform the PCR reactions using the conditions shown on the screen. After the second PCR reaction, combine the four samples into one micro-centrifuge tube. And add 80 microliters of 6X loading dye to it.Visualize the gel and confirm a band at approximately 250 base pairs. Human MPNST cells transduced with a non-target control and lentivirus, expressing four different Bcl-6 short hairpin RNA, revealed that several of the Bcl-6 short hairpin RNAs markedly reduced cell numbers. The immuno blot showed that the degree of decrease in cell numbers correlated with the degree of Bcl-6 knockdown.

Cytometer Assays of MPNST Cells Challenged with Therapeutic Agents

To begin, plate the 80%confluent malignant peripheral nerve sheath tumor or MPNST cells at a density of 1200 cells per well in black-walled 96 well plates. After preparing dilutions of the drug to be tested, add each dilution or vehicle to at least three replicate cells. To assess direct cell number, add hoaxed 33342, to a final concentration of one to five micrograms per milliliter to the plates.And incubate the plates for 30 minutes at 37 degrees Celsius. Place the plates on a high throughput imaging cytometer and scan the plates using the direct cell count for total cell number option with 100, 000 to 600, 000 milliseconds exposure times. Analyze the reads using the software, export them into a spreadsheet and use appropriate software for statistical analyses.