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

Screening op genoomschaal om therapeutische doelen in tumoren te identificeren en te vergelijken

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.

Auteur in de schijnwerpers: nieuwe therapeutische doelen vinden voor kwaadaardige perifere zenuwschedetumoren door middel van shRNA-schermen op genoomschaal 

Genetische profilering en drop-outscreening op genoomschaal om therapeutische doelen te identificeren in muismodellen van kwaadaardige perifere zenuwschedetumoren

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  

Lentivirale verpakking en virale titering voor shRNA-schermen op genoomschaal

Op dag nul plaat 10 miljoen 293T-cellen in 30 milliliter antibioticavrije DMEM met 10% FBS in een schaaltje van 15 centimeter. Bereid in totaal 10 van dergelijke gerechten. Bevestig de volgende dag, de eerste dag, dat de cellen voor 80% confluent zijn en klaar voor transfectie.Voeg vervolgens in een conische buis van 50 milliliter achtereenvolgens 600 microliter van 0,5 microgram per microliter verpakkingsplasmidemix en 60 microliter plasmide-barcodebibliotheek toe. Meng in een aparte conische buis van 15 milliliter 900 microliter transfectiereagens en 12 milliliter DMEM door middel van vortexen. Voeg 12,9 milliliter van dit mengsel toe aan de DNA-mix en veeg gewoon om te combineren.Zonder verder te mengen, incubeer het mengsel gedurende 15 minuten op kamertemperatuur. Voeg vervolgens druppelsgewijs 2,5 milliliter van het geïncubeerde mengsel toe aan elke schaal van 15 centimeter met de 293T-cellen en incubeer de cellen een nacht in een weefselkweekincubator. Oogst het virus op dag drie door de media door een filtratie-eenheid van 0,2 micron te halen.Aliquot het filtraat dat virale deeltjes bevat in conische buizen van 15 milliliter. Bereid bovendien vijf aliquots van één milliliter gefilterd virus in cryoflacons voor virale titering. Om de lentivirale pools te titeren, voegt u 65 microliter kationisch polymeer toe aan een steriele glazen kolf met 65 milliliter tumorcelgroeimedia.Pipetteer één milliliter van het polymeer bevattende media per putje in 11 weefselkweekplaten met zes putjes. Probeer vervolgens de tumorcellen in de vroege passage te proberen. Nadat u de cellen hebt geteld met behulp van een voorkeursmethode, brengt u de cellen over naar de platen met zes putjes.Ontdooi de aliquots van één milliliter van het 48-uurs lentivirus uit de vriezer in een waterbad van 37 graden Celsius. Volg de tabel om de infectie voor elke virale module voor te bereiden.

Research

JoVE Journal

Cancer Research

Lentiviral Packaging and Viral Titering for Genome-Scale shRNA Screens

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

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