BackgroundHCMV encodes a stable 5 kb RNA of unknown function that is conserved across cytomegalovirus species. In vivo studies of the MCMV orthologue, a 7.2 kb RNA, demonstrated that viruses that do not express the RNA fail to establish efficient persistent replication in the salivary glands of mice. To gain further insight into the function and properties of this conserved locus, we characterized the MCMV intron in finer detail.MethodsWe performed multiple analyses to evaluate transcript expression kinetics, identify transcript termini and promoter elements. The half-lives of intron locus RNAs were quantified by measuring RNA levels following actinomycin D treatment in a qRT-PCR-based assay. We also constructed a series of recombinant viruses to evaluate protein coding potential in the locus and test the role of putative promoter elements. These recombinant viruses were tested in both in vitro and in vivo assays.ResultsWe show that the 7.2 kb RNA is expressed with late kinetics during productive infection of mouse fibroblasts. The termini of the precursor RNA that is processed to produce the intron were identified and we demonstrate that the m106 open reading frame, which resides on the spliced mRNA derived from precursor processing, can be translated during infection. Mapping the 5′ end of the primary transcript revealed minimal promoter elements located upstream that contribute to transcript expression. Analysis of recombinant viruses with deletions in the putative promoter elements, however, revealed these elements exert only minor effects on intron expression and viral persistence in vivo. Low transcriptional output by the putative promoter element(s) is compensated by the long half-life of the 7.2 kb RNA of approximately 28.8 hours. Detailed analysis of viral spread prior to the establishment of persistence also showed that the intron is not likely required for efficient spread to the salivary gland, but rather enhances persistent replication in this tissue site.ConclusionsThis data provides a comprehensive transcriptional analysis of the MCMV 7.2 kb intron locus. Our studies indicate that the 7.2 kb RNA is an extremely long-lived RNA, a feature which is likely to be important in its role promoting viral persistence in the salivary gland.
Development of the next generation of anti-cancer drugs, biologics, and immunotherapies is currently hampered by extremely poor success rates of seemingly promising experimental therapies in human clinical trials. This poor success rate is partially due to a lack of biologically relevant cancer model systems. The 2D culture system, while relatively inexpensive and easy to use, may not always be adequately representative of the tumor microenvironment. Conversely, the use of animal models is costly and time consuming. The 3D culture system is able to represent biologically relevant complexities such as cell-cell communication, differential proliferation rates, and compound penetration. Combining 3D culture with CRISPR/Cas9 genome editing technologies can generate powerful cell-based model systems with both physiological relevance and well-controlled genetic and drug-susceptibility profiles. These new models will enable the investigation of specific molecular mechanisms, biofunctional outcomes of newly identified genetic alternations, and targeted therapeutic drug responses within a more biologically intricate context. In this study, we use CRISPR/Cas9 to generate isogenic drug-resistant melanoma models that can be used as either 2D or 3D cancer models, as well as for studies of the acquired drug resistance. Two different models were generated starting with the BRAF-inhibitor sensitive A375 melanoma cell line. We introduced either the NRASQ61K or the KRASG13D point mutations, both of which are known to confer BRAF inhibitor resistance and are commonly encountered in BRAF-resistant tumor samples. We then assessed the susceptibility of these new isogenic lines to BRAF inhibitors in both 2D and 3D model systems. We also determined the specific effect of these point mutations on the RAS-RAF-MAPK signaling pathway, a key component of cell-cycle escape and tumor proliferation. Furthermore, we assessed the impact of these mutations on the expression of Programmed Death-Ligand 1 (PD-L1), which recent advances in cancer immunology have directly linked to cancer immune evasion and poor clinical outcomes. Our results show that A375 melanoma isogenic cells carrying KRASG13D have dramatically increased EGFR expression levels, while isogenic cells carrying NRASQ61K have constant activation of the MEK-ERK pathway. We also found a significant increase in PD-L1 expression in KRASG13D, but not in NRASQ61K A375 cells, indicating that expression of PD-L1 is directly linked to KRAS mutation or the downstream effects thereof, rather than to a general increase in RAS-RAF-MAPK signaling. These results have direct implications for cancer immune checkpoint molecule studies, and highlight the utility of these isogenic melanoma models in both 2D and 3D applications in research and development of novel anti-cancer drugs and combination therapies. Citation Format: Elizabeth Turner, Luping Chen, Johnathan Haag, Alexei Miagkov, Lysa-Anne Volpe, Metewo Selase Enuameh, Robert Newman, Fang Tian. Assessment of RAS-RAF-MAPK pathway perturbations and PD-L1 expression in an Isogenic 3D tissue-culture model of drug-resistant melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5021.
How to balance high efficiency of transgene expression with low cytotoxicity is a central concern for cell biology. Developing lipid-based transfection reagents that have those attributes and can be conveniently used for multiple cell types is critical for studies in cancer biology and cell biology. To address these concerns, ATCC has developed two lipid-based transfection reagents, GeneXPlus and TransfeX™ that provide a powerful tool for efficiently transfecting a broad spectrum of cell types including continuous cancer cell lines, stem cells, immortalized cells, and primary cells with little or no cytotoxicity. For recognized hard-to-transfect cancer cell lines, like THP-1, SH-SY5Y, and Raw 264.7 cells, we have achieved over 30% GFP expression without obvious cytotoxicity by using the GeneXPlus and an EF1α-GFP vector. TransfeX™ has been found to be highly suitable for the transfection of stem cells, immortalized cell lines, and many types of primary cells such as mammary, renal and bronchial epithelial cells, dermal microvascular endothelial cells, and human dermal fibroblasts. We have developed optimized transfection protocols for the TransfeX™ transfection reagent for different cell types and achieved over 50% transfection efficiency and low cytotoxicity. ATCC will provide data showing the broad spectrum of cell types successfully transfected with either GeneXPlus or TransfeX™. Citation Format: Dezhong Yin, Joy Wells, James Clinton, Lysa-Anne Volpe, Kevin Grady, Jeanmarie Curley. Development of lipid-based transfection reagents for efficient expression of transgenes in hard-to-transfect cell types. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-248. doi:10.1158/1538-7445.AM2014-LB-248
Mutant BRAF gene can lead to uncontrolled cell growth through overactivation of RAS-RAF-MAPK signaling pathway. BRAF V600E mutation occurs in approximately 40% to 50% of melanomas. Although BRAF inhibitors have been used to successfully treat melanomas containing the BRAF V600E mutation, patients often become resistant to BRAF inhibitors within a few months. A number of studies have indicated that secondary mutations such as NRAS or NF1 are significantly associated with BRAF resistance by sequencing patient samples. However, due to the genetic heterogeneity commonly observed in tumors, it is unclear if those secondary mutations already existed within low percentage subclones or if they were acquired through drug treatment. It's yet to be determined whether such genetic variants are only associated with resistance, or they actually cause the resistance. In this study, we used genome editing CRISPR technology to generate a drug resistant mutation NRAS Q61K within A375 melanoma cell line which naturally contains BRAFV600E. When compared to the parental line, this isogenic cell model demonstrated that genetically modified NRAS gene at the endogenous level directly leads to a significant resistance to BRAF inhibitors. Method and results: Single guide RNAs (sgRNAs) were designed and built to guide Cas9 to bind and cut desired regions in the NRAS gene target. Melanoma cell line A375 was co-transfected with the single guide and CRIPSR all-in-one plasmid alongside donor plasmid. Transfected cells were sorted into single cells and expanded for subsequent screening of desired gene mutation events. The introduction of NRAS Q61K mutation in the cells was then confirmed via Sanger sequencing and NGS at the genetic level and transcriptional level. Drug responses to BRAF specific inhibitors and non-specific chemotherapy drugs were compared between A375 NRAS Q61K isogenic cell line and parental cell line in 2D and 3D culture environment. Testing results demonstrated that the isogenic cell line created by CRISPR showed significant resistance to BRAF inhibitor in comparison to the parental control in both 2D and 3D culture environment. In summary, we utilized the CRISPR/Cas9 genome editing platform to target endogenous loci in human cells and create the intended genetic mutation event. This new approach provides direct bio-functional evidence of acquiring a drug resistant gene drives tumor cells survival under targeted therapeutic treatment. Furthermore, unlike conventional drug resistant cell models that have been developed through drug selection, the A375 NRAS Q61K isogenic cell line represents a new type of drug resistant model that contains a defined genetic resistance mechanism. It reserves the permanent and genetically stable resistance characteristics without being maintained in drug selection culture environment. Therefore, it provides a valuable tool for developing next generation therapeutics that can overcome BRAF drug resistant in melanoma. Citation Format: Lysa-Anne Volpe, Metewo Selase Enuameh, Luping Chen, Michael Jackson, Catherine Nguyenngo, John Foulke, Fang Tian. New type of drug resistant isogenic cancer cell model created by CRISPR genome editing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3836. doi:10.1158/1538-7445.AM2017-3836
Melanoma remains the most lethal form of skin cancer exhibiting high mortality rates due to a high likelihood of developing metastases and acquiring drug resistance. Approximately 40-50% of melanomas contain oncogenic BRAF mutations of which 75-90% harbor the BRAFV600E mutation. This mutation constitutively activates the mitogen-activating protein kinase (MAPK) signaling pathway leading to uncontrolled cell growth and oncogenesis. Recent combination therapies of BRAF and MEK specific inhibitors have shown improved progression-free patient response in phase II clinical trials. However, in the majority of patients, acquired resistance to MAPK pathway inhibitor therapies develops after approximately 12 months of treatment. Preclinical studies have suggested that MEK1 mutations confer resistance to BRAF and MEK inhibitors. In this study, we used the CRISPR genome editing technology to generate a drug resistant MEK1Q56P knock-in mutation within the A375 melanoma cell line which naturally harbors the BRAFV600E mutation. We validated this new isogenic cell model using both molecular and biofunctional approaches. Method and results: Single guide RNAs (sgRNAs) were designed and built into Cas9 plasmids to bind and cut desired regions in the MEK1 target region. The melanoma cell line A375 was co-transfected with single guide-containing Cas9 plasmids and donor plasmids. Single cells were cloned and expanded for subsequent screening of the desired gene mutation events. The introduction of the MEK1Q56P mutation in the cells was then confirmed via Sanger sequencing and NGS at the genetic level. Validation of the mutation in mRNA transcripts was carried out by RT-PCR followed by sequencing. The cell line maintains permanent and genetically stable resistance characteristics during cell culture expansion without the use of selective pressure. Drug responses to BRAF and MEK1 specific inhibitors and non-specific chemotherapy drugs were compared between the A375 MEK1Q56P isogenic cell line and the parental cell line in 2D and 3D culture environments. Results demonstrated that the isogenic MEK1Q56P cell line showed significant and specific resistance to BRAF inhibitors in comparison to the parental A375 line. Using gene editing we have targeted an endogenous loci within a melanoma cell line creating a novel in-vitro model to aid in basic and translational melanoma research as well as drug screening efforts. This new approach to cell line development provides direct in vitro bio-functional evidence of a drug resistant gene driving tumor cells survival under targeted anti-cancer treatments. Furthermore, this A375 MEK1Q56P isogenic cell line represents a new type of drug resistance model that contains a defined genetic resistance mechanism. This model provides an invaluable tool for developing next generation therapeutics that can overcome drug resistance in melanoma. Citation Format: Lysa-Anne Volpe, Metewo Selase Enuameh, Luping Chen, Elizabeth Turner, John Foulke, Fang Tian. Development of a novel MEK mutation driven drug resistant melanoma model by CRISPR/Cas9 technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2153.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.