lentiviral packaging and viral titering for genome-scale shrna screens

종양의 치료 표적을 식별하고 비교하기 위한 게놈 규모 스크리닝

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.

저자 스포트라이트: 게놈 스케일 shRNA 스크리닝을 통한 악성 말초 신경초 종양에 대한 새로운 치료 표적 찾기 

악성 말초 신경초 종양의 마우스 모델에서 치료 표적을 식별하기 위한 유전자 프로파일링 및 게놈 규모 드롭아웃 스크리닝

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  

게놈 스케일 shRNA 스크리닝을 위한 렌티바이러스 패키징 및 바이러스 적정

0일차에는 15cm 접시에 10% FBS를 함유한 30밀리리터의 무항생제 DMEM에 1,000만 개의 293T 세포를 담았습니다. 총 10 가지 요리를 준비합니다. 다음 날인 첫째 날에 세포가 80% 융합되어 transfection할 준비가 되었는지 확인합니다.그런 다음 50 밀리리터 원뿔형 튜브에 600 마이크로리터의 0.5 마이크로그램의 마이크로리터 포장 플라스미드 믹스와 60 마이크로리터 플라스미드 바코드 라이브러리를 순차적으로 추가합니다. 별도의 15 밀리리터 원뿔형 튜브에 900 마이크로리터의 transfection 시약과 12 밀리리터의 DMEM을 볼텍싱으로 혼합합니다. 이 혼합물 12.9 밀리리터를 DNA 혼합물에 넣고 튕겨서 결합하십시오.더 이상 혼합하지 않고 혼합물을 실온에서 15분 동안 배양합니다. 그런 다음 2.5T 세포가 들어 있는 각 15cm 접시에 배양된 혼합물 2.5mL를 293mL를 넣고 조직 배양 인큐베이터에서 밤새 배양합니다. 3일째에는 0.2미크론 여과 장치를 통해 배지를 통과시켜 바이러스를 채취합니다.바이러스 입자를 포함하는 여과액을 15 밀리리터 원뿔형 튜브에 분취합니다. 또한 바이러스 역정을 위해 극저온 바이알에 여과된 바이러스의 1밀리리터 분취액 5개를 준비합니다. 렌티바이러스 풀의 역가를 높이려면 65mL의 종양 세포 성장 배지가 들어 있는 멸균 유리 플라스크에 65μL의 양이온성 폴리머를 추가합니다.웰당 1mL의 폴리머 함유 배지를 11개의 6웰 조직 배양 플레이트에 피펫팅합니다. 다음으로 초기 통과 종양 세포를 트립신화합니다. 선호하는 방법을 사용하여 세포를 계수한 후 세포를 6웰 플레이트로 옮깁니다.냉동실에서 48시간 렌티바이러스 1밀리리터 분취액을 섭씨 37도의 수조에서 해동합니다. 표에 따라 각 바이러스 모듈에 대한 감염을 준비합니다.

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Cancer Research

Lentiviral Packaging and Viral Titering for Genome-Scale shRNA Screens

143 Views.  Medical University of South Carolina. 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 microlite...